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

Preservation of fish male germplasm in Poland

The natural resources of a country, including ichthyofauna, constitute a vital aspect of its national heritage. Fish populations are threatened with loss of biodiversity as a result of human activity (anthropopressure), resulting in water pollution, habitat destruction and overfishing. Additionally, the escalating threat is exacerbated by climate change, primarily manifested in periodic reservoir and watercourse desiccation. Genetic variability of captive is also threated as fish raised in hatcheries are susceptible to bacterial and viral diseases. Therefore, methodologies for fish sperm cryopreservation aimed at safeguarding the gene pool of both natural and captive fish populations assume paramount importance for their conservation and mitigation of irreversible losses, particularly crucial in light of increasing ecological disasters. This paper offers an overview of cryopreservation research in Poland, tracing back to early initiatives in the 1970s concerning carp (Cyprinus carpio) semen and culminating in recent advancements, where standardized cryopreservation methodologies were developed. We delve into the freezing results of semen of various fish species, encompassing both wild specimens like whitefish (Coregonus lavaretus) and lake minnows (Eupallasella percnurus), and farmed species such as sturgeons, carp, and numerous salmonid species. Additionally, we delineate projects that support such endeavors. Recent milestones in the establishment of fish sperm cryobanks in Poland catering to both wild and farmed species, including carp and rainbow trout (Oncorhynchus mykiss) – the most economically significant fish in Poland were presented. We also expound on the implementation of cryopreserved semen from sex-reversed rainbow trout in hatchery practices. Furthermore, we discuss significant challenges pertaining to sperm banking, particularly concerning funding and the practical utilization of cryostored semen samples for egg fertilization under hatchery conditions

Characterization of carp seminal plasma Wap65-2 and its participation in the testicular immune response and temperature acclimation

International audienceTwo functionally distinct isoforms of warm-temperature acclimation related 65-kDa protein (Wap65-1 and Wap65-2) with a role in the immune response are present in fish. To our knowledge, contrary to Wap65-1, Wap65-2 has neither been isolated nor functionally characterized in carp especially in reproductive system. The aim of this study was to characterize Wap65-2 and ascertain its functions in immune response and temperature acclimation within reproductive system. Wap65-2 corresponded to one of the most abundant proteins in carp seminal plasma, with a high immunologic similarity to their counterparts in seminal plasma of other fish species and a wide tissue distribution, with predominant expression in the liver. The immunohistochemical localization of Wap65-2 to spermatogonia, Leydig cells, and the epithelium of blood vessels within the testis suggests its role in iron metabolism during spermatogenesis and maintenance of blood-testis barrier integrity. Wap65-2 secretion by the epithelial cells of the spermatic duct and its presence around spermatozoa suggests its involvement in the protection of spermatozoa against damage caused by heme released from erythrocytes following hemorrhage and inflammation. Our results revealed an isoform-specific response of Wap65 to temperature acclimation and Aeromonas salmonicida infection which alters blood-testis barrier integrity. Wap65-2 seems to be related to the immune response against bacteria, while Wap65-1 seems to be involved in temperature acclimation. This study expands the understanding of the mechanism of carp testicular immunity against bacterial challenge and temperature changes, in which Wap65-2 seems to be involved and highlights their potential usefulness as biomarkers of inflammation and temperature acclimation

Secreted novel AID/APOBEC-like deaminase 1 (SNAD1) – a new important player in fish immunology

The AID/APOBECs are a group of zinc-dependent cytidine deaminases that catalyse the deamination of bases in nucleic acids, resulting in a cytidine to uridine transition. Secreted novel AID/APOBEC-like deaminases (SNADs), characterized by the presence of a signal peptide are unique among all of intracellular classical AID/APOBECs, which are the central part of antibody diversity and antiviral defense. To date, there is no available knowledge on SNADs including protein characterization, biochemical characteristics and catalytic activity. We used various in silico approaches to define the phylogeny of SNADs, their common structural features, and their potential structural variations in fish species. Our analysis provides strong evidence of the universal presence of SNAD1 proteins/transcripts in fish, in which expression commences after hatching and is highest in anatomical organs linked to the immune system. Moreover, we searched published fish data and identified previously, “uncharacterized proteins” and transcripts as SNAD1 sequences. Our review into immunological research suggests SNAD1 role in immune response to infection or immunization, and interactions with the intestinal microbiota. We also noted SNAD1 association with temperature acclimation, environmental pollution and sex-based expression differences, with females showing higher level. To validate in silico predictions we performed expression studies of several SNAD1 gene variants in carp, which revealed distinct patterns of responses under different conditions. Dual sensitivity to environmental and pathogenic stress highlights its importance in the fish and potentially enhancing thermotolerance and immune defense. Revealing the biological roles of SNADs represents an exciting new area of research related to the role of DNA and/or RNA editing in fish biology

Cryoprotectant-specific alterations in the proteome of Siberian sturgeon spermatozoa induced by cryopreservation

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods—liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability

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