[Show abstract][Hide abstract] ABSTRACT: Artificial propagation of sturgeons is becoming increasingly important for recovery efforts as well as for commercial production. Sterlet Acipenser ruthenus is a common Eurasian sturgeon with a small body size and one of the fastest reproductive cycles among the sturgeons. The practical question being addressed in this study was how long fertilization of ovulated eggs can be delayed without substantially reducing the hatching rate, and an ancillary question, under what temperature conditions do eggs retain good quality. Broodstock were injected with homogenized carp pituitary extract (CPE); ovulated eggs from three females were allocated to various treatment groups for temperature storage (control, 7, 11, 15, & 19 °C) until fertilized. Storage times at the regulated temperatures prior to fertilization were for 2.5, 5.0, 7.5 & 10.0 h. After the selected storage times in ovarian fluid, pooled eggs were fertilized and transferred to incubation cages and neurulation were counted. Three replicates were allocated to each storage period and temperature. Hatched larvae were counted at 7 d post-fertilization. We found that sterlet eggs do not need to be fertilized immediately after collection. Reasonably good quality was retained for several hours if temperature conditions are fairly cool and stable. Eggs retained good quality when stored at 7 and 11 °C for up to 10 h with 54.1 ± 2.9 to 69.9 ± 7.9% hatching success, but egg quality was significantly reduced after 5 h storage at 19 °C (P < 0.01) and 7.5 h storage at 15 °C (P < 0.05) compared to cooler temperatures. Uniform temperatures between 7 and 11 °C can be considered as appropriate for storage of eggs in ovarian fluid for up to 10 h. This information can have practical application to routine hatchery practice for acipenserids, as well as for certain research protocols.
Full-text · Article · Jan 2016 · Reproduction in Domestic Animals
[Show abstract][Hide abstract] ABSTRACT: Sturgeon spermatozoa maturation during their passage through the kidney is a prerequisite for initiation of motility. Samples of sterlet () testicular sperm (TS) were matured in vitro by incubation in seminal fluid (SF) or in SF supplemented with carbonyl cyanide -chlorophenyl hydrazone (CCCP; a respiration uncoupling agent). Sperm was diluted in activation medium (AM) containing 10 m Tris-HCl buffer (pH 8.5) and 0.25% Pluronic, and spermatozoon motility was assessed. Samples were taken and fixed in 3 perchloric acid at 3 points in the incubation process. Quantification of ATP, ADP, and creatine phosphate (CrP) was conducted using liquid chromatography/high-resolution mass spectrometry. We observed a significant decrease in CrP during artificial maturation of TS in SF. In contrast, ATP and ADP were not significantly affected. Addition of CCCP to SF halted maturation and led to significantly lower CrP whereas ADP significantly increased and ATP was unaffected. Dilution of matured and immature TS with AM led to a significant decrease of ATP and CrP and an increase of ADP compared with their levels before dilution, although immature TS were not motile. Energy dependency of TS maturation in sturgeon was confirmed, which suggests that mitochondrial oxidative phosphorylation is needed for maturation of sturgeon TS.
No preview · Article · Dec 2015 · Journal of Animal Science
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Nov 2015 · Fish Physiology and Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Spermatozoa are stored in a quiescent state in the male reproductive tract and motility is induced in response to various environmental stimuli, such as change of osmolality (general case) and a decrease of extracellular K(+) in fish from Acipenseridae family. This study was aimed to investigate the relationship between osmolality and extracellular K(+) concentration in controlling sperm motility in sturgeon. Pre-incubation of sturgeon sperm for 5s in hypertonic solutions of glycerol, NaCl, or sucrose (each of 335mOsm/kg osmolality) prepares sturgeon spermatozoa to become fully motile in presence of high concentration of K(+) ions (15mM), which has previously been demonstrated to fully repress motility. Furthermore, presence of 0.5mM KCl during the high osmolality pre-incubation exposure completely prevented subsequent spermatozoa activation in a K(+)-rich media. Manipulating the transport of K(+) ions by the presence of K(+) ionophore (valinomycin), it was concluded that once an efflux of K(+) ions, the precursor of sturgeon sperm motility activation, is taking place, spermatozoa then become insensitive to a large extracellular K(+) concentration.
Full-text · Article · Nov 2015 · Animal reproduction science
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The egg structure of sturgeon is complex, with multiple micropyles and an envelope structure different from that of teleosts and other fish groups. In the sturgeon, an adhesive layer in the follicular epithelium is responsible for the egg adhesiveness. This is a problem for artificial reproduction of many fish species, including sturgeon, when a large number of eggs are incubated in hatchery conditions. Although several techniques for removing adhesiveness have been developed and successfully applied to eggs of common carp, tench, pikeperch and European catfish, de-sticking methods for sturgeon are limited. Proteolytic enzymes have been successfully applied to common carp, tench and African catfish eggs, leading to a hatching rate over 80% following 2-min treatment. In sturgeon, blue clay is the most commonly used de-sticking substance. The chemical reactions determining egg adhesiveness in sturgeon are poorly studied, and optimum methods for removing stickiness remain to be developed.
Full-text · Article · Jul 2015 · Reviews in Aquaculture
[Show abstract][Hide abstract] ABSTRACT: Three groups of seven pond-cultured pikeperch males, held under controlled conditions of low water temperature and for a varying photoperiod were injected with 500 IU kg−1 human chorionic gonadotropin. Following a latent period of 72 h, sperm was collected. Stripping was on 26 March (Group A), 21 April (Group B), and 13 June (Group C). Spermatozoa was obtained from 85% fish in group B and from 42% of fish in group C. Mean volume of stripped semen for Group A was 0.64 ml, for Group B 1.07 ml, and for Group C 1.80 ml, while the mean concentration of spermatozoa was similar in all groups (15.73 ± 2.68–19.34 ± 3.87 109 ml−1). Group A spermatozoa showed the longest motile period (89.93 ± 10.20 s) and Group B the shortest (55.18 ± 10.46 s). The highest velocity at 15 s post-activation was recorded in group A (220 ± 22.3 μm s−1) and the lowest in Group B (159 ± 35 μm s−1). Group C showed velocity of 187 μm s−1. The results of our study showed that the length of the cold water period had no influence on spermatozoa quality, but did have an influence on the ability of males to produce sperm.
Full-text · Article · Jun 2015 · Journal of Applied Ichthyology
[Show abstract][Hide abstract] ABSTRACT: The objective of this study with common carp spermatozoa was to understand the fertilization potency of different males when different sperm quantities were applied per ova of a single female. The sperm of five males representing very good sperm motility and the ova from one female exhibiting the best apparent quality were used. The sperm of each male was collected at volumes of 5 (1000 spermatozoa), 10 (5000 spermatozoa), 20 (10 000 spermatozoa), 40 (20 000 spermatoza) and 400 μl (200 000 spermatozoa) and pre-diluted with 995, 990, 980, 960 and 600 μl of Kurokura solution, respectively. Thereafter, 4000 eggs and pre-diluted spermatozoa from each male, one by one, were simultaneously added to 1000, 5000, 10 000, 20 000 and 200 000 spermatozoa and activated with hatchery water. Initial sperm motility was in the range of 89.5-97.2% at 15 s, decreasing to 19.1-30.2% at 60 s post-activation. At all times of evaluated post-activation, the sperm motility did not differ significantly among the males. Sperm velocity decreased from 126.1 to 161.2 μm s−1 at 15 s to 11.9-35.2 μm s−1 at 60 s post-activation. Sperm velocity was significantly different among males at 15 s post-activation. Fertilization and hatching rates were similar in all males at a higher examined number of spermatozoa per ova (20 000 and 200 000). Similar fertilization and hatching rates were observed in four out of five males at 10 000 spermatozoa per ova. Lower spermatozoa per ova (5000) induced very different results, from 48 to 82% for fertilization rates and from 42 to 72% for hatching rates. At 1000 spermatozoa per ova a very high variability was observed: 10-50% for fertilization rates and 8-43% for hatching rates. These results did not correspond to sperm velocity among males. The 20 000 spermatozoa density was considered as providing a secure number of spermatozoa for reaching good fertilization in common carp. To avoid loss of genetic variability for future generations this recommendation is important to know for the management of hatcheries where these broodstocks will be generated.
No preview · Article · Jun 2015 · Journal of Applied Ichthyology
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A practical technique for isolation and cryopreservation of tench (Tinca tinca) (Cyprinidae, Teleostei) early stages of germ cells (GC), including spermatogonia and spermatocytes, is reported for the first time. The germ-line cells possess the ability to differentiate into functional gametes of both sexes. These early stages of germ cells are small enough to be well-suited to cryopreservation, which, together with their high level of plasticity, makes their preservation a promising tool for maintaining genetic resources. Testicular cells were distinguished and separated by Percoll gradient, with the highest proportion of GC (62.2%) obtained from the 30% layer. The concentration and viability of GC were determined, and specific staining (DDX4) for germ cells was used to distinguish GC from somatic cells. Early stages of germ cells were cryopreserved in an extender composed of phosphate buffered saline (pH 8) with 0.5% BSA, 50mM d-glucose, and containing 1.5M cryo-protectant in the pre-programmed PLANER Kryo10 series III using a cooling protocol from +10°C to –80°C at a rate of 1°C/min. The effect of six cryoprotectants – methanol, dimethyl sulfoxide, dimethyl sulfoxide + propanediol (1 : 1), glycerol, ethylene glycol, and dimethylacetamid was assessed, and the results were evaluated by comparing the percentage of viable frozen/thawed GC by ANOVA, Tukey's HSD test (P < 0.05). Almost the same viability rates were obtained with no significant differences among tested cryoprotectants, indicating high stability of GC in cryoprotectants. Nevertheless, glycerol at a concentration of 1.5M was associated with the highest survival rate of thawed tench GC (57.69 ± 16.85%).
Full-text · Article · Aug 2014 · Czech Journal of Animal Science
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jul 2014 · Fish Physiology and Biochemistry