Sylwia Judycka

New extender for cryopreservation of Siberian sturgeon (Acipenser baerii) semen

The goal of this study was to develop a simple glucose-methanol extender for cryopreservation of Siberian sturgeon (Acipenser baerii) semen. Semen quality was assessed by determining post-thaw sperm motility and fertilizing ability at hatching stage. We tested the effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30 M) in a methanol extender on post-thaw sperm motility. Sperm motility parameters and fertilizing ability of semen cryopreserved in 0.1 M glucose in 15% methanol (GM) were compared to previously described Tris-sucrose-KCl in 10% - methanol extender (TSKM). Additionally, sperm motility and fertilizing ability in relation to 30 min equilibration in GM extender before cryopreservation and 30 min of post-thaw storage were determined. The beneficial effect of the glucose for semen cryopreservation was related to its concentration with a quite narrow optimum of 0.1 to -0.15 M. The fertilization rates of frozen/thawed sperm were similar for both (TSKM and GM) tested extenders. The sperm motility and fertilization rate were not affected either by 30 min equilibration in GM extender or by 30 min of post-thaw storage. Our work indicates that the use a simple extender consisting of 0.1M glucose in 15% methanol can be an alternative cryopreservation method to those previously described for sturgeons. The use of an equilibration period and the possibility of post-thaw semen storage can improve organization of hatchery work and help with logistics of large-scale hatchery operations.

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.

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.

The effect of different ambient temperatures on river lamprey (Lampetrafluviatilis) egg and sperm production under controlled conditions

Reproductive performance (ovulation/spermation rate and relative fecundity of females) of adult river lamprey (Lampetrafluviatilis) was compared among adults held under three different controlled thermal regimes (7, 10 and 14°C). The quantity of semen (volume of semen, sperm concentration, total sperm production and total number of sperm) and the weight of the eggs as well as the semen quality (sperm motility, seminal plasma osmolality and pH, sperm pH and total protein content) were determined. Housing temperature had no apparent effect on quality or quantity of eggs produced, but did influence time of ovulation. On the other hand, temperature had a significant effect on the quantity and quality of sperm produced; 70% of males held at 10°C and 14°C did not spermiate. The best ambient temperature for river lamprey adults held under controlled conditions was 7°C. All males kept in this temperature yielded mature semen with the highest sperm motility parameters. It was also found that among the six tested solutions, the most suitable artificial medium for river lamprey sperm activation was 20mM Tris buffer containing 40mM NaHCO3 at pH 8.5 and 100mOsmkg-1.

Androgenetic development of X- and Y-chromosome bearing haploid rainbow trout embryos.

Haploid fish embryos are important in studies regarding role of the recessive traits during early ontogeny. In fish species with the male heterogamety, androgenetic haploid embryos might be also useful tool in studies concerning role of the sex chromosomes during an embryonic development. Morphologically differentiated X and Y chromosomes have been found in a limited number of fish species including rainbow trout (Oncorhynchus mykiss Walbaum 1792). To evaluate role of the sex chromosomes during rainbow trout embryonic development, survival of the androgenetic haploids in the presence of X or Y sex chromosomes has been examined. Androgenetic haploid rainbow trout were produced by fertilization of X-irradiated eggs with spermatozoa derived from the normal males (XY) and neomales, that is, sex-reversed females (XX) to produce X- and Y-bearing haploids, and all X-bearing haploids, respectively. Survival rates of the androgenetic progenies of normal males and neomales examined during embryogenesis and at hatching did not differ significantly. However, all haploids died within next few days after hatching. Cytogenetic analysis of the androgenetic embryos confirmed their haploid status. Moreover, apart from the intact paternal chromosomes, residues of the irradiated maternal chromosomes observed as chromosome fragments were identified in some of the haploids. Provided results suggested that rainbow trout X and Y chromosomes despite morphological and genetic differences are at the early stage of differentiation and still share genetic information responsible for the proper embryonic development.

Substrate specificity of proteolytic activity in the testes fluid and seminal plasma of the common carp Cyprinus carpio.

Substrate specificity in the seminal plasma and testes fluids of the common carp Cyprinus carpio was determined using gelatin, casein, albumin and haemoglobin. Proteolytic profiles of the testes and seminal plasma were compared. Different ranges of pH (5·5-9·5) and temperature (4-37° C) were used during incubations of seminal plasma proteinases. Differences in proteolytic activity between testes and seminal plasma may reflect specific functions of the testes and sperm ducts in semen production. Seminal plasma metalloproteinases were characterized by higher substrate specificity than were serine proteinases. Zymography optimization for seminal plasma indicated that pH 7·5 and 22° C were the optimal conditions for gel incubations.

Effects of different stripping methods of female and activation medium on fertilization success in northern pike (Esox lucius)

In this study, the quality of northern pike eggs collected by the traditional method (hand stripping) and the pneumatic method (air stripping) was compared. Additionally, different activation solutions (Billard solution, hatchery water, and Woynarovich solution) were tested for their effects on egg fertilization under artificial conditions. After the eggs were collected, the Pseudo-Gonado-Somatic Index (PGSI) was measured. Although the values of the PGSI in the samples obtained with use of air stripping were lower (13.8 ± 3.9%), they did not differ statistically from those obtained by hand stripping (16.5 ± 5.4%). Hatchery water and Woynarovich solution were found to be the most suitable solutions for sperm activation using the ComputerAssisted Sperm Analysis (CASA) system as compared to the Billard solution. Hand stripping was found to negatively affect the percentage of fertilized eggs and the percentage of hatched larvae in samples fertilized in hatchery water and Woynarovich solution. When the traditional method of egg collection was used, there were no differences in the percentage of fertilization and the percentage of hatched larvae between Billard solution (54.0 ± 21.5% and 44.1 ± 21.9%, respectively), hatchery water (60.0 ± 22.5% and 55.9 ± 22.8%, respectively), and Woynarovich solution (72.0 ± 25.8% and 69.0 ± 23.9%, respectively) treatments. Air stripped eggs showed a higher fertilization rate when hatchery water or Woynarovich solution was applied (86.2 ± 9.3% and 92.4 ± 3.9%, respectively). Also hatching rate was the highest in these samples (83.0 ± 8.4% and 88.3 ± 6.2%). The application of the pneumatic method and Woynarovich solution for northern pike artificial fertilization resulted in higher fertilization and hatching rates as compared to other techniques. Because this was successful in northern pike, the use of air stripping is a promising option for artificial reproduction in other fish species.

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.

Identification of oxidatively modified proteins due to cryopreservation of carp semen1

During semen cryopreservation, spermatozoa are exposed to physical and chemical stressors that result in their functional and structural damage. Growing evidence suggests that most cryoinjuries result from oxidative stress accompanying sperm cryopreservation. Elevated amounts of reactive oxygen species (ROS) generated during cryopreservation can react with sperm macromolecules, including proteins. The goal of this study was to investigate the oxidative modifications (measured as carbonylation level changes) of carp spermatozoa proteins triggered by the cryopreservation process. Flow cytometry and computer-assisted sperm analysis were used to evaluate changes in viability, ROS level, and motility of spermatozoa. The spermatozoa proteins that were specifically carbonylated were identified and quantified by Western blotting, in conjunction with 2-dimensional electrophoresis (2D-oxyblot) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Cryopreservation decreased spermatozoa motility (P < 0.01) and viability (P < 0.0001) and significantly increased (P < 0.0001) the number of ROS-positive cells. We identified 25 protein spots, corresponding to 19 proteins, with increases (P < 0.05) in carbonylation level due to freezing/thawing. The identified proteins are involved in motility, metabolism, calcium-ion binding, signal transduction, protein folding, and intracellular transport. The results suggest that carbonylation of flagellar proteins can result in motility disorders and may contribute to the reduced percentage of motile spermatozoa and disturbances in movement trajectory after sperm cryopreservation. Moreover, cryopreservation may contribute to impaired cellular respiration, ATP regeneration, disturbances of Ca2+ turnover, unfolding of cytoplasmic or histone proteins, disturbances of cell signaling and intracellular transport, and reduced membrane stability. Our results contribute to the knowledge concerning cryoinjury and to further development of a modified cryopreservation procedure aimed at minimizing oxidative damage of carp sperm proteins.

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.