Artificial reproduction of wild and pond-cultured common tench (Tinca tinca) under controlled conditions.

The common tench is an important thermophilic freshwater fish used for aquaculture diversification. This species is farmed in ponds as well as caught in the wild in many European and China. Artificial reproduction under controlled conditions is one of the most important part of modern aquaculture. In this study, two experiments were conducted in which the influence of hormonal agents (Ovopel and Ovaprim - experiment I) and the origin of the spawning stock (wild and pond-cultured - experiment II) on the success of reproduction in terms of ovulation rate (%), latency time (h), pseudo-gonadosomatic index (PGSI, %), hatching rate (%), abnormal larvae rate (%), and spawning efficiency coefficient (Se) were examined. While the wild fish could not reproduce in the absence of hormonal treatment with 0 % ovulation rate. In all tested hormonal treatment allowed to obtain high-quality eggs with hatching rates of over 83 %. Significant differences in latency time were observed among groups from different origins, such as differences in ovulation rate (79 % vs. 90 %, for pond-cultured and wild fish, respectively), PGSI (8.2 vs. 8.9), and hatching rate (63.8 % vs. 84.3 %), which showed significantly better quality in fish from the wild than those from pond culture. This means that when spawners of common tench of various origins are available, it is better to choose spawners from natural waters for artificial reproduction.

Effect of dilution in sperm maturation media and time of storage on sperm motility and fertilizing capacity of cryopreserved semen of sex-reversed female rainbow trout.

Masculinized females, also called neomales or sex-reversed females have a male phenotype but retain the female genotype (XX). Therefore, all spermatozoa produced in their functional testes carry an X chromosome, which is desired for the production of all-female rainbow trout populations. Semen of sex-reversed female rainbow trout is of low quality and in vitro maturation is required, which includes dilution of sperm suspensions with specially formulated maturation solutions. The aim of this study was to determine the effect of dilution in different maturation media on sperm quality (sperm motility characteristics and fertilizing capacity) of frozen/thawed sperm of sex-reversed female rainbow trout. The effect of time of post-thaw storage (0, 15, 60 and 120min) on semen quality was also tested. Sperm motility parameters and fertilization rate at the eyed and hatching stages were assessed for post-thaw semen diluted in different media. The cryopreservation procedure resulted in high post-thaw sperm motility of about 57% and did not differ from fresh semen. Unexpectedly, maturation media decreased sperm activation capacity immediately after dilution; however, sperm motility increased over time. Fertilization rates of frozen/thawed semen were high (71-87%) and did not differ significantly between experimental variants at any of tested periods of storage. Our results demonstrated that the effect of the maturation media on frozen/thawed sperm is different from that of fresh sperm. The progressive increase in post-thaw sperm motility in maturation media can potentially be applied to routine hatchery practice.

Gonadogenesis and annual reproductive cycles of an endangered cyprinid fish, the lake minnow Eupallasella percnurus (Pallas, 1814).

In this study, gonadogenesis, the effect of temperature (15, 20 and 25°C) on sex differentiation, and annual changes in the gonads of mature lake minnow Eupallasella percnurus (Pallas, 1814) were determined. The lake minnow was found to be a primary gonochoristic fish species, where gonads are formed directly in the ovaries or testes. The morphological differentiation of gonads was initiated 35days post hatch (DPH) when two types of gonadal anlages were visible: a pear-shaped gonad attached by a single mesentery string and a spindle-shaped gonad attached on both sides to the peritoneum. Gonadogenesis occurred faster in females than in males, with the first previtellogenic oocytes and ovarian lamellae being already observed in 45 DPH fish. In males, cytological differentiation occurred approximately 85 DPH, when the fish reached an average body weight of more than 400mg. No significant effect of rearing temperature on sex ratio in lake minnow juveniles was observed. The proportion of males and females was similar (close to 1:1) in all of the thermal-treated groups, although there were effects of temperature on the final sizes of fish. Histological examination of wild, mature lake minnow ovaries during the annual cycle (from May to February the following year) showed asynchronous oocyte maturation. The testes were characteristic of multi-batch spawning fish. Quantitative dominance of spermatids and mature spermatozoa in May was observed, while the presence of primary and secondary spermatocytes in all other periods was confirmed. These changes were also reflected in the seasonal variation in the gonado-somatic index in both sexes, with the highest mean values of 11.2% (females) and 4.0% (males) in May, which were found to be significantly different to all other periods. The data presented in this study provide an important contribution to our understanding of the biology and reproductive strategy of the endangered lake minnow.

Motility of carp spermatozoa is associated with profound changes in the sperm proteome.

In freshwater cyprinids, spermatozoa are quiescent in seminal plasma and sperm motility is initiated by a decrease in osmolality (hypo-osmotic shock) after discharge into the aqueous environment. However, it is unknown at present if and to what extent changes in proteins are involved in carp sperm motility. Therefore, the aim of our study was to identify proteins related to carp sperm motility through a comparison of immobilized and activated carp spermatozoa using a 2D-DIGE approach. Our results, for the first time indicated that carp sperm motility is associated with changes in protein content. Seventy-two differentially expressed proteins were identified. These proteins are mainly involved in ubiquitin-proteasome pathways, glycolysis, the TCA cycle, remodeling and are putatively related to sperm energy metabolism and motility. Moreover proteins associated with oxidative stress responses, signal transduction by Ca(2+)-dependent MAPK cascades, and PKC and protein folding have been identified. The proteins involved in carp sperm motility were localized to the cytoplasm, mitochondria, cytoskeleton, nucleus and sperm membrane. The identification of a high number of proteins involved in carp sperm motility would contribute to current knowledge about the molecular mechanisms of sperm motility in freshwater fish. BIOLOGICAL SIGNIFICANCE: To the best of our knowledge, few changes in proteins involved in the initiation of fish sperm motility have been identified. This is a limited number of proteins compared with the 80 recently identified proteins involved in human sperm motility. However, no proteomic studies of sperm motility have yet been performed on freshwater fish. Our present study allowed for the first time a comprehensive characterization of the proteins associated with carp sperm motility and a better understanding of the molecular mechanisms underlying sperm motility activation and maintenance. The application of 2D-DIGE facilitated the identification proteins crucial for sperm structural organization and motility. The identification of a high number of proteins involved in carp sperm motility would contribute appreciably to the presently limited information available on the mechanisms of sperm motility in freshwater fish. Moreover the identified list of proteins will create a platform for future studies designed to assess the functional significance of specific proteins in sperm motility.

Temporal Changes in Gametogenesis of the Invasive Chinese Pond Mussel Sinanodonta woodiana (Lea, 1834) (Bivalvia: Unionidae) from the Konin Lakes System (Central Poland).

Gametogenesis and the temporal changes occurring in the ovaries and testes throughout the reproductive cycle in the invasive alien bivalve, Chinese pond mussel Sinanodonta woodiana (Lea), from the heated Konin lakes system (central Poland) were studied using histological techniques. S. woodiana was confirmed to be a gonochoristic species with overall sex ratio of 1:1. The examined morphological parameters of Chinese pond mussel spermatozoa, i.e. 42 µm mean total length; 4.3 µm mean head length and the maximum size of previtellogenic (34-43 µm) and vitellogenic oocytes (75-83 µm) are consistent with values established for closely related members of the Unionidae family. Our results suggest that S. woodiana in the Konin lakes system are able to spawn throughout March to October, with a season of higher reproductive activity in females extending from March to April. This type of reproductive biology may contribute to the Chinese pond mussel's success in thriving in freshwater ecosystems.

Proteomic analysis of extracellular medium of cryopreserved carp (Cyprinus carpio L.) semen.

During freezing and thawing, spermatozoa are exposed to physical and chemical stressors that result in adverse changes in sperm structures and physiological functions. The present study provides, for the first time, a comprehensive description of protein changes in the extracellular medium of cryopreserved semen. Using 2D-DIGE and a combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, 183 proteins released from sperm to an extracellular medium were identified. The majority of released proteins were involved in metabolism and energy production. Moreover, proteins associated with a response to stress, apoptosis, small GTPase mediated signal transduction, transcription, translation, protein folding and turnover, reproduction and DNA repair were identified. The dominant group of released proteins was related to cytoplasm. Moreover, specific proteins associated with the membrane, mitochondria and nucleus were identified. The identification of a high number of proteins released from sperm provides new insight into the mechanism of cryodamage to the particular sperm structure and to specific metabolic pathways, which were affected by cryopreservation. The availability of a catalog of carp sperm proteins altered by cryopreservation provides a crucial tool for the development of novel potential biomarkers of cryoinjuries and for the improvement of a long-term sperm preservation procedure.

Maturation of spermatozoa from rainbow trout (Oncorhynchus mykiss) sex-reversed females using artificial seminal plasma or glucose-methanol extender.

Masculinized females (sex-reversed females) produce only homogametic spermatozoa (X) for fertilization which is desired for the production of all-female rainbow trout populations. The milt of sex-reversed females is of low quality and must be matured through extension in maturation solutions. The aim of this study was to compare the usefulness of glucose-methanol (GM) extender with artificial seminal plasma (ASP) extender for the maturation of milt of sex-reversed female rainbow trout. Milt suspensions were incubated at 4 °C for either 15 minutes (GM extender) or 120 minutes (ASP extender). Incubation of milt diluted in either the GM or ASP extender caused a significant (P < 0.05) increase in the percentage of sperm motility to 76.1 ± 10.9% and 74.7 ± 18.6% for GM and ASP, respectively, but no differences between both the extenders were found. Incubation also increased the average path velocity, straight line velocity, and linearity values of spermatozoa diluted with the GM extender; at the same time, none of the other parameters changed for ASP suspensions. Sperm diluted with ASP was characterized by higher curvilinear velocity and lateral head displacement values. Percentage of eyed embryos produced by fertilization using milt diluted in the GM extender amounted to 63.6 ± 16.4% and 67.2 ± 11.9% for sperm-to-egg ratio of 300,000:1 or 600,000:1, respectively and was lower (P < 0.05) compared with that of ASP extender (79.5 ± 5.8% and 80.3 ± 4.7% for sperm-to-egg ratio of 300,000:1 or 600,000:1, respectively). The results of our study clearly report that the mechanism of sperm maturation by the GM extender differs from that based on ASP.

Biochemical and physiological characteristics of semen of sex-reversed female rainbow trout (Oncorhynchus mykiss, Walbaum).

This works studies the biochemical (protein concentration, osmolality, antitrypsin activity, lactate dehydrogenase activity) and physiological characteristics (sperm motility characteristics) of semen of sex-reversed female rainbow trout (n=42) obtained with the application of 11ß-hydroksyandrostendione for sex reversal. All data were arbitrarily divided into three classes depending on the percentage of sperm motility: I XX<25%; II XX 25-50% and III XX>50%. The average percentage of sperm motility was 18±7% n=12 (group I XX); 42±6% n=15 (group II XX) and 65±12% n=15 for group III XX, respectively) to link the values of semen parameters to the maturation stage of semen. Semen from 12 normal males of the same age was used as a reference group. Sperm concentration as well as protein concentration, osmolality, antitrypsin activity, and lactate dehydrogenase activity in seminal plasma of sex-reversed females were higher compared with the values obtained for normal male rainbow trout. The values of these parameters declined with the increasing percentage of sperm motility toward values established for normal males. The fertilization success of semen (3×10(6) spermatozoa/egg) of sex-reversed females was very high (above 90%) for both the percentage of eyed embryos and hatched larvae and was related to sperm motility classes. Correlations between the quality parameters of sex-reversed females semen corresponded to those established previously for the semen of normal male rainbow trout. Antitrypsin activity, lactate dehydrogenase, protein concentration, and osmolality were found to be characteristic of seminal plasma of sex-reversed females. The maturity of sex-reversed female spermatozoa seems to be associated with the decline in the values of those parameters toward the values characteristic for seminal plasma of normal males.

Carp transferrin can protect spermatozoa against toxic effects of cadmium ions.

Cadmium is a widespread heavy metal that enters the aquatic environment and affects many processes involved in fish reproduction such as sperm motility. Fish seminal plasma proteins can protect spermatozoa against toxic effects of heavy metals. The objective of this study was to demonstrate the ability of a major carp seminal plasma protein-transferrin (TF) to bind cadmium ions and to neutralize the toxic effect of cadmium on carp sperm motility. To obtain a high quantity of carp seminal plasma TF necessary for the experiment, immunoaffinity chromatography as a one-step isolation procedure was established. The titration of TF with cadmium ions spectrophotometrically at 247nm revealed that TF binds cadmium ions at only one spectrophotometrically-sensitive binding site, which suggests that TF is capable of neutralizing the cadmium toxic effect. Indeed, the addition of carp TF to carp semen incubated with 50ppm cadmium for 48h led to about a four-times higher percentage of sperm motility (30.3±1.1%) in comparison to samples incubated with only 50ppm cadmium (8.2±5.2%). Similarly, higher values of other parameters of sperm movement measured by a computer-assisted sperm motility analysis system (VSL, VCL and ALH) were observed at the presence of transferrin. In conclusion, our study provides the first evidence that transferrin from carp seminal plasma can protect sperm motility from cadmium toxicity.

Exposure of rainbow trout milt to mercury and cadmium alters sperm motility parameters and reproductive success.

In the current work, seminal plasma was used for the first time as an incubation medium for monitoring short-time exposure effects of sublethal concentrations of mercury and cadmium ions on rainbow trout sperm. Sperm motility parameters (CASA) and hatching rates were used as gamete quality markers. Additionally live/dead sperm viability test and comet assay of DNA fragmentation were performed. We demonstrated that computer-assisted sperm motility analysis (CASA) may serve as a predictor of reproductive success, when milt contaminated with heavy metals is used. Results presented in this study demonstrate that mercury ions altered sperm motility characteristics at 1-10 mg Hg2+/l and 10 mg Cd2+/l and hatching rates at 10 mg Hg2+/l and 10 mg Cd2+/l after 4h of exposure. Although mercury ions affected sperm motility parameters immediately after dilution with milt as well as at 4h of exposure, no differences in sperm motility parameters were found between intact and mercury-treated milt after 24h of exposure. Our results suggest that rainbow trout seminal plasma has a protective role against the toxic effects of mercury ions of rainbow trout sperm motility.

Semen biology of vendace (Coregonus albula L.).

The objective of this study was to describe the morphometry and motility parameters of vendace (Coregonus albula) spermatozoa. Morphometric parameters of vendace sperm head and tail were of values similar to rainbow trout. The effects of pH, sodium, potassium and calcium ion concentrations on computer-assisted sperm analysis (CASA) sperm motility characteristics were tested. Vendace sperm was motile in a wide pH range of 6.0-10.5 with the optimum pH established at 9.0. Increases in potassium and calcium ions caused decreases in the percentage of motile sperm. The CASA parameters and erratic sperm movement pattern of vendace spermatozoa were similar to whitefish (C. lavaretus) sperm motility, suggesting that there is a coregonid-specific sperm motility pattern.

Acrosome staining and motility characteristics of sterlet spermatozoa after cryopreservation with use of methanol and DMSO.

In this study we describe acrosome staining and motility characteristics of fresh and cryopreserved sterlet (Acipenser ruthenus L.) spermatozoa using soybean trypsin inhibitor-Alexa conjugate fluorescent staining and computer-aided sperm analysis (CASA), respectively. Methanol or dimethylsulfoxide (DMSO) were used as cryoprotectants. After cryopreservation a decline in sperm motility characteristics occurred, but no differential effect between cryoprotectant was observed. Cryopreservation caused a significant increase in the percentage of spermatozoa with acrosome stained by SBTI-Alexa for samples cryopreserved using DMSO compared to methanol. These data suggest that the low usefulness of DMSO for cryopreservation of sturgeon spermatozoa is related to its harmful specific effect towards the acrosome, probably by causing its precocious triggering, much before any egg contact.

Effects of different surfactants on motility and DNA integrity of brown trout (Salmo trutta fario) and common carp (Cyprinus carpio) spermatozoa.

In this study we evaluated effects of surfactants on motility parameters and DNA integrity of spermatozoa of freshwater teleost fish. Common carp (Cyprinus carpio) and brown trout (Salmo trutta fario) spermatozoa were exposed to either sodium dodecyl sulphate (SDS, anionic surfactant) or octoxynol 9 ( Triton X-100, nonionic surfactant). Both surfactants added at activation caused a decrease in sperm motility characteristics measured by computer-assisted sperm analysis (CASA). Intraspecific differences in speed and trajectory of movement were detected. Triton X-100 and SDS when added to non activated sperm were also effective in the decrease of sperm motility and caused an increase of DNA fragmentation. Our results suggest that not only sperm motility apparatus but also DNA are targets for surfactant action. Therefore any exposure of spermatozoa to surfactants, in aquaculture conditions or natural environment, would have a negative impact on fish reproduction.

Polymorphism of transferrin of carp seminal plasma: relationship to blood transferrin and sperm motility characteristics.

Transferrin (Tf) is a major protein of carp (Cyprinus carpio) seminal plasma. Its relationship with milt quality is unknown. In this study, we sought to determine if Tf is polymorphic in carp seminal plasma and if this polymorphism is related to sperm motility characteristics. We screened males of purebred common carp line (Polish line R6) for Tf polymorphism in blood plasma. The majority of Tf genotypes represented only DD and DG variants. We then collected milt from preselected DD and DG genotypes and tested their sperm motility characteristics using computer-aided sperm analysis (CASA). Tf polymorphism in seminal plasma was found to be identical with that of blood. However, the relationships between Tf polymorphism and iron metabolic parameters were different for blood and semen. These data suggest different regulation of Tf in liver and testis. We found substantial differences in sperm motility characteristics between both genotypes. Spermatozoa of DG males were characterized by lower curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), higher linearity (LIN) and straightness (STR) of movement as compared to DD males. No differences were found in other sperm characteristics such as sperm concentration and percentage of sperm motility. Our results suggest that sperm motility parameters are related to Tf polymorphism and therefore this polymorphism may be related to sperm competitive ability.