Proteomic analysis of pikeperch seminal plasma provides novel insight into the testicular development of domesticated fish stocks.

Control of the reproduction of domesticated stocks is considered a prerequisite for aquaculture development of pikeperch. However, knowledge about the physiology of the captive pikeperch male reproductive system and the biology of semen is very limited, especially regarding protein characteristics. The aims of our study were to characterize pikeperch sperm quantity and quality parameters and to analyze changes in the proteome of the same males spawned for the first and second times. Moreover, attempts were made to generate the first proteomic library of seminal plasma proteins. Semen collected during the first spawning season was characterized by lower sperm concentration and volume than for the second season. Using mass spectrometry-based label-free quantitative proteomics, we identified 850 proteins in the seminal plasma of pikeperch from both spawning seasons, and 65 seminal proteins were found to be differentially abundant between the first and second spawning seasons. The majority of differentially abundant proteins were involved in stress and immune responses, developmental processes, cofactor metabolic processes, proteolysis, cellular oxidant detoxification and organization of the extracellular matrix (ECM). In addition, several proteins unique to pikeperch seminal plasma were identified, including antifreeze proteins, hibernation-specific plasma proteins, lectins and vitellogenin. In summary, our results indicate that males that spawned for the first time were characterized by incompletely mature gonads and the expression of proteins associated with the early phase of spermatogenesis and ECM organization. On the other hand, males that spawned for the second time exhibited advanced gonadal maturation and expression of proteins related to the late stage of spermatogenesis and sperm maturation, including regulation of reactive oxygen species generation, bicarbonate production, sperm elongation and separation. The identification of a large number of seminal plasma proteins provides a valuable resource for understanding the functions of seminal plasma and the molecular mechanisms involved in testicular development and maturation in domesticated fish, which is a prerequisite for better control of reproduction in captivity.

Maladaptation of trout spermatozoa to fresh water is related to oxidative stress and proteome changes.

Rainbow trout sperm are 'maladapted' to freshwater spawning, resulting in shorter duration of sperm motility in fresh water compared to buffered saline solution. We hypothesized that different sperm motility-activating media have various effects on sperm motility characteristics and oxidative stress, as well as on the protein profiles of rainbow trout sperm. We designed an experimental model for activation of rainbow trout sperm motility in different osmotic conditions: (i) isosmotic and (ii) hypoosmotic. Spermatozoa activation with hypoosmotic solution was associated with lower values for sperm motility parameters (52%) and an induced increase in ROS level (19.4%) in comparison to isosmotic activation with isosmotic solution (67 and 9.5% for sperm motility and ROS, respectively). Hypoosmotic activation resulted in a higher number of differentially abundant sperm proteins (out of which 50 were identified) compared to isosmotic conditions, where only two spots of protein disulfide-isomerase 6 were changed in abundance. The proteins are mainly involved in the TCA cycle, tight and gap junction signaling, Sertoli cell-Sertoli cell junction signaling and asparagine degradation. Our results, for the first time, indicate that during hypoosmotic activation of sperm motility, osmotic stress triggers oxidative stress and disturbances mostly to structural proteins and metabolic enzymes. Our results strongly suggest that comparative physiological and biochemical analysis of rainbow trout sperm characteristics in isosmotic and hypoosmotic conditions could be a useful model for studying the mechanism of sperm activation in salmonid fish.

Mitochondrial membrane potential and reactive oxygen species in liquid stored and cryopreserved turkey (Meleagris gallopavo) spermatozoa.

The extensive use of artificial insemination in turkeys has led to the development of in vitro semen storage. However, fertility rates from liquid stored and frozen/thawed turkey semen are still unsatisfactory. The aim of the study was to assess spermatozoa viability, mitochondrial membrane potential (MMP), and reactive oxygen species production (ROS) in liquid stored and cryopreserved turkey semen with the use of flow cytometry. Moreover, motility and adenosine triphosphate (ATP) content in sperm were monitored at the same time to link flow cytometry data with sperm movement and energetics. Liquid storage led to a decrease in sperm motility (80.6 vs. 55.6%, for fresh and stored for 48 h), live sperm with an intact MMP (59.9 vs. 30.5% for fresh and stored for 48 h), and a 20-fold decrease in ATP content after 24 h of storage. A 3-fold increase in ROS+ sperm was observed after 48 h of storage (9.3 vs. 26.8% for fresh and stored for 48 h). Semen equilibration before cryopreservation affected only ATP content. However, freezing/thawing led to a dramatic decrease in all of the studied semen quality parameters. A 5-fold decrease in live sperm with intact MMP (59.8 vs. 11.9%) and a 7-fold increase in sperm ROS+ (10.8 vs. 74.4%) were recorded between fresh and frozen/thawed semen. The results strongly suggested that a significant loss of MMP and a disturbance in sperm ATP production during semen storage can be the main reason for the decline in sperm motility. The disturbance of mitochondria activity during storage seems to be associated with the increase in oxidative stress in turkey semen. Turkey sperm mitochondria also appear to be very sensitive to cryodamage. Diminished energy production in turkey spermatozoa, visible as the low percentage of sperm with an intact MMP and low level of ATP after freezing/thawing, which is associated with high ROS generation, could be responsible for the low fertilizing ability of cryopreserved turkey semen.

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.

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.