[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.
Reviews in Aquaculture 07/2015; Early View. DOI:10.1111/raq.12070 · 3.92 Impact Factor
[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.
Journal of Applied Ichthyology 06/2015; DOI:10.1111/jai.12853 · 0.87 Impact Factor
[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.
[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%).
[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.
Fish Physiology and Biochemistry 07/2014; 40(6). DOI:10.1007/s10695-014-9963-2 · 1.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of environmental ion composition and osmolality in calcium ion (Ca2+) signalling of spermatozoa activation over the course of the spawning period of brook trout Salvelinus fontinalis was investigated. Motility at the end of spawning was low (mean ± s.d. of 5 ± 2% motile spermatozoa with curvilinear velocity of 25 ± 8 µm s−1). Addition of 10 mM Ca2+ to the activation medium resulted in values similar to those recorded during the middle of the spawning period (mean ± s.d. of 95 ± 6% motile spermatozoa with curvilinear velocity of 130 ± 15 µm s−1).
Journal of Fish Biology 06/2014; 85(3). DOI:10.1111/jfb.12445 · 1.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of environmental ion composition and osmolality in Ca(2+) signaled activation was assessed in spermatozoa of brook trout Salvelinus fontinalis. Milt from ten mature males was obtained by abdominal massage. Spermatozoa motility was evaluated in 0, 100, and 300 mOsm/kg NaCl or sucrose solutions, buffered by 10 mM Tris-HCl pH 8.5. For investigation of spermatozoa reaction to external Ca(2+) concentration, 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the activation media as a calcium ions chelator. For investigation of the effect of external Na(+) concentration in conditions of low external Ca(2+), 100 µM amiloride was added to the EGTA-containing solutions as a Na(+) transport blocker. Low motility was observed in sucrose (Na(+) free) solutions containing 2 mM EGTA but not in Na(+) solutions containing 2 mM EGTA. Addition of amiloride led to significantly increased motility (P < 0.05) compared with sucrose (Na(+) free) solutions containing 2 mM EGTA. We conclude that Na(+) transport in Ca(2+)-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na(+) and Ca(2+) transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.
Fish Physiology and Biochemistry 04/2014; 40(5). DOI:10.1007/s10695-014-9936-5 · 1.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to examine sperm maturation in sturgeon and to establish the localization of the maturation. We demonstrated that sperm maturation occurs in sturgeon outside the testes via dilution of sperm by urine. The process involves the participation of high molecular weight (>10 kDa) substances and calcium ions.
[Show abstract][Hide abstract] ABSTRACT: Evolution of sturgeons and paddlefishes (order Acipenseriformes) is inherently connected with polyploidization events which resulted in differentiation of ploidy levels and chromosome numbers of present acipenseriform species. Moreover, allopolyploidization as well as autopolyploidization seems to be an ongoing process in these fishes and individuals with abnormal ploidy levels were occasionally observed within sturgeon populations. Here, we reported occurrence of Siberian sturgeon (Acipenser baerii) male with abnormal ploidy level for these species, accessed its ploidy level and chromosome number and investigate its potential sterility or fertility in comparison with normal individuals of sterlet (A. ruthenus), Russian sturgeon (A. gueldenstaedtii) and Siberian sturgeon (A. baerii).
Acipenser ruthenus possessed 120 chromosomes, exhibiting recent diploidy (2n), A. gueldenstaedtii and A. baerii had ~245 chromosomes representing recent tetraploidy (4n), and A. baerii male with abnormal ploidy level had ~ 368 chromosomes, indicating recent hexaploidy (6n). Genealogy assessed from the mtDNA control region did not reveal genome markers of other sturgeon species and this individual was supposed to originate from spontaneous 1.5 fold increment in number of chromosome sets with respect to the number most frequently found in nature for this species. Following hormone stimulation, the spontaneous hexaploid male produced normal sperm with ability for fertilization. Fertilization of A. baerii and A. gueldenstaedtii ova from normal 4n level females with sperm of the hexaploid male produced viable, non-malformed pentaploid (5n) progeny with a ploidy level intermediate to those of the parents.
This study firstly described occurrence of hexaploid individual of A. baerii and confirmed its autopolyploid origin. In addition to that, the first detailed evidence about fertility of spontaneous hexaploid sturgeon was provided. If 1.5 fold increment in number of chromosome sets occurring in diploids, resulted triploids possess odd number of chromosome sets causing their sterility or subfertility due to interference of gametogenesis. In contrast, 1.5 fold increment in number of chromosome sets in naturally tetraploid A. baerii resulted in even number of chromosome sets and therefore in fertility of the hexaploid specimen under study.