Ide (Leuciscus idus) sperm short-term storage: Effects of different extenders and dilution ratios.

In cyprinids, Tyrode's (TLP) and Volckaert's (VRT) solutions are the most frequently used extenders for short-term sperm storage. The effectiveness of TLP and VRT on ide (Leuciscus idus) sperm short-term storage was analyzed using a × 4 (sperm:extender) dilution ratio over 48 h. A × 4 (1:3) dilution ratio was compared to a × 10 (1:9) dilution ratio for ide sperm storage using TLP supplemented with antibiotics and was tested for a 14-day period. Sperm motility (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity of sperm (VCL, µm s-1), movement linearity (LIN, %), beat cross frequency (BCF, Hz), and the amplitude of lateral head displacement (ALH, µm) were verified using a CASA system. After 48 h, most CASA parameters were significantly higher in TLP compared to VRT. The dilution ratio also had a significant impact (P < 0.0001) on the efficiency of ide sperm short-term storage over 14 d in comparison to undiluted sperm (control). Ide sperm diluted with TLP supplemented with penicillin/streptomycin and stored short term, regardless of the dilution ratio used, retained motility and fertilization capacity over 14 d at 4 °C. The highest embryo survival rates of 70% and 73% were noted using sperm diluted with TLP at × 4 and × 10 dilution ratios compared to 48% for raw sperm that was not stored short term.

A Comparison of Pneumatic and Hand Stripping of Whitefish (Coregonus lavaretus) Eggs for Artificial Reproduction.

We describe the technique of pneumatic stripping of whitefish (Coregonus lavaretus) eggs with the use of oxygen, nitrogen, and air. Eggs obtained via the traditional method (by pressing the abdominal surfaces) served as a control group. It was established that the gas flow rate during pneumatic stripping should not exceed 0.5 L∙min-1, since higher air flow resulted in increased post-spawning mortality. The pneumatic stripping method of egg collection was no faster than hand stripping; however, the time required per female was more consistent. It was found that the pH of the ovarian fluid obtained during hand and pneumatic stripping was not related to the success rate of fertilization. Pneumatic stripping resulted in a higher quality of collected eggs and a higher and more consistent hatching rate as compared with the hand-stripped samples, regardless of the gas used. The results presented here lead us to recommend the pneumatic method for obtaining eggs from whitefish, since it is a simple, reproducible method and improves the reproductive performance and developmental success of the fish eggs.

Sperm quality in fish: Determinants and affecting factors.

Fish sperm quality assessment is helpful for optimizing production and for monitoring the environmental state. Sperm can be monitored relatively easy and, to date, various analyses have been applied and proven to be helpful in this task. Among them, sperm motility parameters such as sperm speed are one of the main performance traits during assisted fish reproduction. Apart from motility the sperm concentration, volume, and seminal plasma pH and osmolality are also frequently evaluated and are the main sperm quality indicators measured in fish sperm. However, other parameters also determine sperm fertilization potential. Recent knowledge reveals several additional parameters of high importance for sperm function. Among them are DNA integration, membrane stability, mitochondria status and enzymatic activity. Measuring all these parameters in fish sperm provides complex knowledge regarding male fertility and helps to improve broodstock maintenance protocols as well as gamete handling and fertilization processes. This review focuses on the presentation of the sperm quality measures for freshwater and marine species of the fish and provides information regarding recent methods of sperm quality evaluation.

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario).

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

Optimisation of sodium and potassium concentrations and pH in the artificial seminal plasma of common carp Cyprinus carpio L.

The effect of sodium and potassium concentrations as well as optimal pH on the motility of common carp Cyprinus carpio L. sperm during short-term storage in artificial seminal plasma (ASP) was investigated. Sperm was collected from individual males (n = 5) and each sample diluted tenfold (1:9) in ASP (sperm:extender) containing 2 mM CaCl2, 1 mM Mg2SO4 and 20 mM Tris at pH 8.0 and supplemented by the following concentrations of sodium and potassium (mM/mM): 0/150, 20/130, 40/110, 75/75, 110/40, 130/20 and 150/0. The osmolality of all ASP variants was set at 310 mOsm kg-1. Sperm motility was measured using a CASA system during 72 h of storage. Immediately after dilution, sperm motility was high (90%) both in each variant and in the control group (fresh sperm). After 72-h storage, the highest sperm motility was noted in ASP containing 110 mM NaCl and 40 mM KCl. No differences were found in the motility of samples preserved within the pH range of 7.0-9.0. Our data suggest that for the short-term storage of common carp sperm, whereas the pH of the solution does not play a crucial role, a specific potassium concentration of around 40 mM is required.

Multiple collections of common carp Cyprinus carpio L. semen during the reproductive period and its effects on sperm quality.

Multiple collections of semen during the reproductive period of the common carp Cyprinus carpio L. were used to analyse changes in semen quality. Semen collection was performed on June 1 (first collection), 12 (second collection), and 19 (third collection) from individual males (n=11) by gentle abdominal massage. Semen quantity (semen volume and sperm count), quality (sperm motility and sperm viability), as well as seminal plasma parameters (pH of seminal plasma and seminal plasma osmotic pressure) and its enzymatic activity, e.g., lactate dehydrogenase (LDH) and ß-N-acetylglucosaminidase (ß-NAG) were determined. Moreover, for the first time, the percentage of live, dead, and apoptotic sperm, as well as the proteolytic activity of seminal plasma, were determined using flow cytometry and zymography, respectively, at specific times during the common carp reproductive period. The lowest volumes of semen and sperm concentration were noted during the first semen collection (June 1). Analysis of computer-assisted sperm analysis parameters revealed the greatest sperm motility, sperm velocity, as well as amplitude of lateral head displacement, were evident in the third collection (June 19). There were no differences in progressively motile sperm, movement linearity, wobbling index, and beat cross frequency between the different collection times. The lowest percentage of live sperm was found in the first collection, although with the passage of time values of this parameter increased. Seminal plasma pH and seminal plasma osmotic pressure were at the lowest values in the second collection (June 12), which corresponded with the lowest concentration of sperm. In the first collection, seminal plasma contained the highest values of LDH and ß-NAG activity, whereas there were no differences in the proteolytic activity of seminal plasma determined between the different collections of semen. The results presented here indicate that during the reproductive period, males of common carp produce a large amount of semen of moderate quality. Low sperm motility noted in the second collection might be explained by a significant increase in sperm production during this period, followed by a low seminal plasma pH and high hydration rate. The high LDH and ß-NAG activity noted in the first collection of semen may reflect a reduced stability of the sperm cell membrane and its viability. The significant difference in the percentage of live sperm at June 1 compared to that at June 19 supports this hypothesis.

Serine-like proteolytic enzymes from common carp Cyprinus carpio L. seminal plasma are able to degrade sperm proteins.

The application of zymography, with sperm proteins as a substrate, allowed for the first time the visualisation of two serine proteinases with a molecular weight of 76 and 163kDa from common carp Cyprinus carpio L. seminal plasma. Twenty four hours of incubation in a development solution with a pH of 7.5 and incubation at 37°C were the best conditions for the visualisation of serine proteinase; however, proteolysis was also observed at 4°C. Our results indicate that serine proteinase from common carp seminal plasma with a molecular weight of 76 and 163kDa may be involved in the degradative mechanism of sperm proteins. This mechanism may be responsible for the removal of damaged sperms by the digestion of native sperm proteins.

Evidence of the sterility of allotetraploid Cobitis loaches (Teleostei, Cobitidae) using testes ultrastructure.

The diploid-polyploid populations of Cobitis distributed in Poland are usually composed of the spined loach Cobitis taenia or, less often, the Danubian loach C. elongatoides and their triploid (females) and tetraploid hybrids (females and males). The aim of this study was to determine whether tetraploid males participate in the reproduction process by analyzing their testis ultrastructure and the process of spermatogenesis in comparison with diploid males of both parental species. Tetraploid loaches were obtained from three different diploid-polyploid populations distributed in Poland. The structure of Cobitis testes are typical for most Teleostei fish with cystic-type spermatogenesis. The successive stages of developing germ cells are enclosed within cysts formed by the Sertoli cells. This paper morphologically describes the different germ cell stages of spermatogenesis (spermatogonia, spermatocytes, spermatids, and spermatozoa) of C. taenia and C. elongatoides and provides a pioneering ultrastructural analysis of tetraploid Cobitis testes which reveals their unusual structure for the first time. Thus, cysts with normal spermatogonia and spermatocytes (pachyten or leptoten stages) containing synaptonemal complexes were present and no spermatids or spermatozoa were observed. Moreover, in contrast to previously analyzed diploid species, single cells or all of the cells within the cysts displayed chromatin condensation and/or chromatin fragmentation. The obtained results clearly demonstrated that tetraploid males are sterile and diploids are fertile and are the only sperm donors in the reproduction processes of diploid-polyploid Cobitis populations.

Cryopreservation of Acropora digitifera sperm with use of sucrose and methanol based solution.

Coral biodiversity has recently been considered an important topic in environmental studies. Biodiversity could be preserved with successful cryopreservation of endangered species gametes or embryos. Herein, we developed cryopreservation protocols for Acropora digitifera sperm with use of sucrose and methanol based extender. We studied cryopreservation of A. digitifera sperm with floating frames, allowing the placement of 250 µl French straws 4 cm above the liquid nitrogen surface, resulting in a 40 °C/min freezing rate. This method enabled the successful cryopreservation of sperm in 0.9 M sucrose supplemented with 20% methanol. In this protocol, we used a 1:3 (sperm:extender) dilution ratio. The fertilization ratios of freezing:thawed sperm were similar to the control and reached 63%. This method might be a valuable option in the formation of A. digitifera gene banking. Further studies are needed to explore possibilities of using this method in cryopreservation of other coral's sperm.

Artificial reproduction of wild and cultured barbel (Barbus barbus, Cyprinidae) under controlled conditions.

The aim of this work was to compare the effects of controlled reproduction of cultured and wild common barbel, Barbus barbus (L.). Preparations containing different GnRH analogues and dopamine receptor antagonists (Ovopel, Ovaprim) as well as human chorionic gonadotropin (hCG) (in the case of cultured fish) were applied and their influence on ovulation, spermiation and quality of gametes obtained was determined. No differences in the qualitative or quantitative parameters of semen were found between fish stimulated with different hormonal preparations and those not receiving hormonal stimulation. The high suitability of Ovaprim for ovulation induction in (cultured and wild) barbel was confirmed. The highest synchronisation of ovulation was obtained after the application of Ovopel (18 ± 3 h), but the best results of controlled reproduction (expressed as the percentage of ovulations and survival of embryos) were obtained by applying Ovaprim (83.2 ± 4.1). A significantly higher percentage of ovulation was obtained in cultured fish (80-90%) than in wild fish (< 25%).