Sperm Production and Cryopreservation in Muskellunge after Carp Pituitary Extract and Human Chorionic Gonadotropin Injection

We investigated the effects of carp pituitary extract (CPE) and human chorionic gonadotropin (hCG) on the sperm production in muskellunge Esox masquinongy. Total volumes of milt collected from fish (mean weight, 4.8 ± 1.5 kg) injected with CPE, hCG, or the saline control were 5.36 ± 3.75 mL, 3.1 ± 1.52 mL, and 3.89 ±2.16 mL, respectively. Sperm concentration, protein and mineral concentrations of semen, and osmolality of seminal plasma were similar in control and hormonally treated fish. Hormonal injections did not affect the initial percentage of motile sperm compared to untreated fish. However, motility of sperm from the CPE group was lower than for the saline group at 75 s after activation (statistical significance was P = 0.06). The fertilizing capacities of spermatozoa after cryopreservation from CPE-injected fish were similar to, if not better than, control fish. We report here, for the first time, the successful cryopreservation of muskellunge semen, which produced 30.1 ± 3.8% survival to the eyed-embryo stage versus 72.9 ± 8.7% survival obtained with fresh semen.This work was funded by Federal Aid in Sport Fish Restoration project F-69-P (Fish Management in Ohio), which is administered jointly by the U.S. Fish and Wildlife Service and the Ohio Division of Wildlife. Salaries were partly provided by state and federal funds appropriated to the Ohio Agriculture and Development Center. This is manuscript 173/95

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 were 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

Proteomic analysis of carp seminal plasma provides insights into the immune response to bacterial infection of the male reproductive system

Aeromonas salmonicida is recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. However, the mechanism of immunity to these bacteria in common carp is still not well understood, especially the immune regulation in the gonad to bacterial infection. The aims of our study were to analyze changes in the seminal plasma proteome following A. salmonicida infection in carp males. The observed pathological changes in the tissue (liver, spleen, kidney and testis) morphology and upregulation of immune-related genes (tnfa2, il6a) confirmed the successful infection challenge. Using mass spectrometry-based label-free quantitative proteomics, we identified 1402 seminal plasma proteins, and 44 proteins (20 up- and 24 downregulated) were found to be differentially abundant between infected and control males. Most differentially abundant proteins were involved in the immune response mechanisms, such as acute phase response, complement activation and coagulation, inflammation, lipid metabolism, cell-cell and cell-matrix adhesion, creatine-phosphate biosynthesis and germ cell-Sertoli cell junction signaling. Bacterial infection also caused profound changes in expression of selected genes in the testis and hematopoietic organs, which contributed to changes in seminal proteins. The altered seminal proteins and bacterial proteins in seminal plasma may serve as valuable markers of infection in the testis

Proteomic characterization of fresh spermatozoa and supernatant after cryopreservation in relation to freezability of carp (Cyprinus carpio L) semen.

Our recent studies suggested that the freezability of carp semen is related to seminal plasma protein profiles. Here, we aimed to compare the spermatozoa proteomes of good (GF) and poor (PF) freezability semen of carp. To achieve this, we used two-dimensional difference in gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry. The semen was classified as GF or PF based on sperm motility after freeze/thawing. We identified proteins enriched in spermatozoa of GF (22 proteins) and PF (18 proteins) semen. We also identified 12 proteins enriched in the supernatant after cryopreservation of PF semen. Good freezability is related to high concentrations of proteins involved in the maintenance of flagella structure, membrane fluidity, efficient control of Ca2+ and sperm motility, energy production, and antioxidative protection, which likely reflects the full maturation status of spermatozoa of GF semen. On the other hand poor freezability seems to be related to the presence of proteins identified as released in high quantities from cryopreserved sperm of PF. Thus, the identified proteins might be useful bioindicators of freezing resilience and could be used to screen carp males before cryopreservation, thus improve long-term sperm preservation in carp. Data are available via ProteomeXchange with identifier PXD008187