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Cryopreservation of Goldlined seabream Rhabdosargus sarba (Forsskål, 1775) sperm: CASA observation and enzyme activity evaluation
Abstract
In this study, we developed an optimized cryopreservation protocol for goldlined seabream sperm through five small experimental groups: Diluting the sperm with a mixed cryoprotectant (containing 10 g/L glucose and 10% DMSO in an RS extender) at a ratio of 1:3, followed by equilibration at 4 °C for 30 min. Subsequently, the samples were subjected to liquid nitrogen steam fumigation at 5 cm above the surface for 5 min before drop in the liquid nitrogen. Using computer-assisted sperm analysis (CASA), we examined the motility parameters of the sperm before and after freezing for 2 days, as well as the changes in energy metabolism enzyme (T-ATP, LDH, SDH) activities and antioxidant enzyme (T-GSH, CAT, SOD, and GSH-px) activities at 0, 5, 10, and 20 days after freezing. After 250 days cryopreservation, fluorescence staining of sperm showed that most cell membranes were less damaged, and the fertilization rate of sperm after 250 days cryopreservation was 72.53 ± 7.32%. objective of this study was to select an effective cryopreservation protocol for goldlined seabream sperm and elucidate the physiological damage mechanisms before and after cryopreservation.
... Seabream is the main type of teleost cultured in coastal areas of China. The annual production of Seabream (such as yellowfin seabream, black seabream, and goldlined seabream) in the years 2020-2022 were 122.5, 131.0, and 136.5 thousand tons respectively (Pan et al. 2024a;Fisheries 2021). The yellowfin seabream Acanthopagrus latus is the main farmed variety in the southern regions of China, known for its good meat quality and strong resilience, with wild populations often found at river mouths and nearshore waters . ...
... Prediction of the signal peptide was achieved through SignalP 5.0. The gene structure of AlGal-8 was elucidated using the GSDS (Hu et al. 2015), and its domain conservation was assessed with the SMART (Pan et al. 2024a). Clustal Omega (Sievers and Higgins 2018) has been used to compare AlGal-8's amino acid sequences with those of Gal-8 from other species, and visualized by JalView (Waterhouse et al. 2009). ...
Seabream is one of most important candidates for aquaculture in China. However, in recent years, streptococcosis has posed a serious challenge. Galectin-8 has been proven to play an important role in the antibacterial immunity of teleost. In this study, Acanthopagrus latus Galectin-8 (AlGal-8) was characterized for the first time consisting of 8 exons and 7 introns. AlGal-8 protein was considered to dock with N-Acetylglucosamine in the CRD region. In healthy tissues, AlGal-8 was found to be expressed highest in the gills, followed by brain, kidney and liver. After infection with Streptococcus iniae, the expression of AlGal-8 in the brain, kidney, and liver increased to varying degrees, but there was no significant change in the spleen. We also analyzed SNPs present in the genomic region of AlGal-8, and a total of 8 SNPs were found. One SNPs in exon (G2264C) and two in introns (T4799G and C4892T) were significantly (P < 0.05) associated with resistance to S. iniae. This study not only provides a reference for the role of Galectin-8 in the antibacterial immunity of teleost but also provides fundamental information for the disease-resistant molecular breeding of A. latus.
... The protein molecules in FBS stabilize the extracellular environment, reducing osmotic damage during freezing and protecting cell membranes through specific interactions with cell surfaces [47]. FBS also provides nutritional support and promotes cell adhesion [48]. Consequently, MET and FBS are more suitable for preserving high bioactivity and cell integrity in sensitive biological samples at high concentrations, whereas other cryoprotectants may be less effective in providing added protection at elevated concentrations [49]. ...
In cryopreservation technology, the choice of cryoprotectant plays a crucial role in cell survival and function. Different types of cryoprotectants, each with unique protective mechanisms, mitigate cellular damage from ice crystal formation during freezing. This study investigated the effects of different types and concentrations of cryoprotectants on the cryopreservation efficacy of noble scallop Mimachlamys nobilis sperm. Six cryoprotectants were tested, including four permeable cryoprotectants (dimethyl sulfoxide (DMSO), ethylene glycerol (EG), propylene glycerol (PG), methanol (MET)) and two non-permeable cryoprotectants (trehalose (TRE), fetal bovine serum (FBS)). The results showed that permeable cryoprotectants, which penetrate the cell membrane, regulate the osmotic pressure inside and outside cells to reduce dehydration damage. Among them, 10% DMSO provided the best protection, significantly preserving sperm motility, velocity, and morphology. Non-permeable cryoprotectants, although unable to penetrate cells, stabilized the extracellular environment at higher concentrations (such as FBS). Additionally, MET and FBS exhibited enhanced protective effects with increasing concentration, indicating their potential in reducing sperm structural damage at higher concentrations. Morphological observations indicated that freezing caused varying degrees of structural damage to sperm, with flagellar integrity being crucial for motility. Overall, selecting an appropriate cryoprotectant and concentration is essential for the efficient cryopreservation of M. nobilis sperm, providing a valuable reference for conserving germplasm resources of marine species.
... Finally, the relevant activities were calculated using a formula. The experimental procedures were carried out according to the manufacturer's instructions and were repeated six times to ensure consistency [34,35]. ...
This work explores the digestive system characteristics of Brachymystax tsinlingensis during early developmental stages and aims to solve the problem of high lethality of fry during the transgression period, which is crucial for the artificial propagation and population conservation of endangered fishes. This study was carried out on intestinal tissue, digestive enzymes, and antioxidant enzyme activities in the early development stage of Brachymystax tsinlingensis. Ten random samples during endogenous nutrition (7, 10, and 11 days after hatching), mixed nutrition (13 and 19 DAH), and exogenous nutrition (31, 33, 39, 45, and 73 DAH) were collected by histological and biochemical analysis methods. The results showed that the intestine of Brachymystax tsinlingensis already has four layers initially at 7 DAH, and the intestinal gland tissue is evident at 73 DAH. The contents of total protein (TP) and the activities of lipase (LPS) and trypsin (TPS) were maximal at 39 DAH, and the activities were 3.20 ± 0.26 mg/mL, 2.52 ± 0.69 U/g, and 2717.45 ± 295.26 U/mg, respectively. Catalase (CAT) and glutathione peroxidase (GSH-PX) activities both showed the lowest values at 39 DAH, which were 0.57 ± 0.11 U/mg and 3.35 ± 0.94 U/mg, respectively. The activity of amylase (AMS) and the content of malonaldehyde (MDA) increased, and the highest values were reached at 45 DAH (1.32 ± 0.41 U/mg) and 73 DAH (1.29 ± 0.43 nmoL/mg), respectively. Superoxide dismutase (SOD) and GSH-PX activities both showed a peak value at 7 DAH (126.58 ± 20.13 U/mg and 6.47 ± 1.86 U/mg). Overall, the changes in intestinal tissue, digestive enzymes, and antioxidant enzyme activities at 39 DAH of Brachymystax tsinlingensis are inseparable from different vegetative stages during the developmental period, and these results can provide a reference for the proliferation and cultivation of Brachymystax tsinlingensis resources.
... Yellowfin seabream Acanthopagrus latus is an excellent candidate for aquaculture in China, known for its tender flesh, strong salinity adaptation, and good resistance to adversity (Liang et al., 2023;Pan et al., 2023) (Liang et al., 2023;Pan et al., 2023Pan et al., , 2024b. However, there have been frequent reports in recent years of yellowfin seabream infections by S. ...
To date, little attention has been paid to identifying the effects of long-term cryopreservation on sperm quality for Piaractus orinoquensis. The object of this study was therefore to evaluate the effect of long-term cryopreser-vation (24 h, 1, 6 and 12 months) on sperm motility, viability, DNA integrity, ATP content, total antioxidant capacity (TAC), morphology and sperm ultrastructure in this species. Milt samples from six males were cry-opreserved in a medium containing final concentrations of 7.5% Me 2 SO, 4.1% glucose and 9.0% egg yolk. The samples were frozen in liquid nitrogen (LN) vapor and stored in LN for periods of 24 h and 1, 6 and 12 months. After thawing, both the motility rate and the viability decreased significantly compared with fresh sperm; however, these parameters did not differ among the four cryopreservation times. The DNA integrity and ATP content decreased significantly after 6 months of cryopreservation. There were no significant differences in TAC values between fresh and cryopreserved sperm. The total sperm abnormalities in cryopreserved samples were about 5-fold higher than in fresh sperm; short tail was the most common defect occurring after cryostorage. The ultrastructural analysis reveals that P. orinoquensis spermatozoa consist of an ovoid head without acrosome, a cylindrical mid-piece, and a single flagellum. The nuclear fossa is located at the base of the nucleus and contains the centriolar complex. There are 1-2 ring-shaped mitochondria located in the mid-piece. The flagellum shows a 9 + 2 organization of microtubules in the axoneme. Post-thaw spermatozoa presented damage such as swelling and rupture of the plasma membrane, mitochondrial damage, loss of the electron-dense chromatin of the nucleus , and degeneration in the middle region.
Mitochondrial health is crucial to sperm quality and male fertility, but the precise role of mitochondria in sperm function remains unclear. SDHA is a component of the succinate dehydrogenase (SDH) complex and plays a critical role in mitochondria. In humans, SDH activity is positively correlated with sperm quality, and mutations in SDHA are associated with Leigh Syndrome. Here we report that the C. elegans SDHA orthologue SDHA-2 is essential for male fertility: sdha-2 mutants produce dramatically fewer offspring due to defective sperm activation and motility, have hyperfused sperm mitochondria, and disrupted redox balance. Similar sperm motility defects in sdha-1 and icl-1 mutant animals suggests an imbalance in metabolites may underlie the fertility defect. Our results demonstrate a role for SDHA-2 in sperm motility and male reproductive health and establish an animal model of SDH deficiency-associated infertility.
Simple Summary
The roughscale sole, Clidoderma asperrimum, is found in the East and West Seas of Korea, waters north of Hokkaido in Japan, as well as the East China Sea, the East Pacific, and the Canadian Maritimes. In 2021, this fish was categorized as an endangered species. Thus, there is a need to maintain gametes by freezing sperm. This study investigated the impacts of the cryoprotective agent, diluent, dilution ratio, and thawing temperature on the cryopreservation of fish sperm. In this investigation, sperm dilution 1:1 with a mixture of 10% dimethyl sulfoxide + Stein’s solution and thawing at 10 °C provided the most effective DNA damage prevention. These results support the development of a roughscale sole sperm cryopreservation procedure.
Abstract
The roughscale sole, Clidoderma asperrimum is categorized as an endangered species. Sperm freezing is essential for preserving gametes. This study examined the CPA concentration, diluent, dilution ratio, and thawing temperature to design a sperm cryopreservation protocol for roughscale sole. The variables examined included sperm motility and kinematics, cell survival, fertilization, and DNA fragmentation. Sperm motility parameters were assessed via computer-assisted sperm analysis using a CEROS II instrument. Cell survival rate and DNA damage were assessed using the Cell Counting Kit-8 and single-cell gel electrophoresis assay, respectively. Sperm preservation was tested using several CPAs, including ethylene glycol, dimethyl sulfoxide (DMSO), glycerol, propylene glycol, and methanol. The diluents tested were 300 mM sucrose, 300 mM glucose, Stein’s solution, Ringer’s solution, and Hank’s solution. The optimal conditions for sperm cryopreservation were 10% DMSO + Stein’s solution. After thawing, sperm motility was highest with a 1:1 dilution ratio (sperm to CPA + diluent), at 69.20 ± 0.32%; thawing at 10 °C was optimal for post-thaw motility (72.03 ± 0.95%). The highest fertilization rate (40.00 ± 1.22%) was obtained using DMSO. The fresh sperm had the lowest tail DNA, followed by 10% DMSO + Stein’s solution. The developed cryopreservation methods can be used in roughscale sole hatcheries.
Although sperm cryopreservation is well developed, different methods can be used for freezing fish semen, mainly with the intention of reducing the use of liquid nitrogen. For this purpose, ultra-freezers could, among other advantages, facilitate the maintenance of semen compared to liquid nitrogen containers. Indeed, ultra-freezers have an increased capacity to store semen, and they can be available in places where there are no other technologies. The aim of this study was to develop an alternative protocol for freezing streaked prochilod Prochilodus lineatus semen using an ultra-freezer. The fish semen (n=10) was collected and frozen using an ultra-freezer (−80°C), with a solution containing 10% DMSO diluted in 5% Beltsville thawing solution in the proportion of 1:4 (semen/diluent) in 0.5-mL straws. Each week, a tube was randomly chosen, thawed, and used to evaluate seminal parameters using computerized semen analysis (CASA). Then, the data were subjected to analysis of variance followed by Tukey’s post hoc test. Motility, motility time, vitality, sperm velocity, and secondary morphology were influenced by storage in ultra-freezer (p < 0.05). However, these parameters were kept at levels considered adequate during the time tested. This is, to the best of our knowledge, the first report of frozen P. lineatus semen under ultra-freezer conditions (−80°C), demonstrating that this method is efficient in maintaining the semen quality for up to 10 weeks.
The marbled flounder (Pseudopleuronectes yokohamae) is a commercial flatfish in East Asia. The aim of this study was to improve its sperm cryopreservation protocol based on the vitality assessment of 7-day and 1-year cryopreserved sperm. Four extenders (extender-1: sucrose solution; extender-2: glucose solution; extender-3: fish Ringer's solution; and extender-4: modified fish Ringer's solution) were tested with a combination of five cryoprotectants (CPAs) (dimethyl sulfoxide: Me2SO; glycerol: GLY; ethylene glycol: EG; propylene glycol: PG; and methanol: MeOH) at four different concentrations (5, 10, 12, and 15%). Fluorescent technique was applied to detect the plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and DNA integrity of fresh and cryopreserved sperm specimens. Fresh sperm was diluted at a ratio of 1:2 (sperm:extender). Post-thaw motility of sperm cryopreserved using 15% Me2SO along with either extender-1 (86.0 ± 5.2%) or extender-2 (85.7 ± 7.1%) was similar (p > 0.05) to that of fresh sperm. Sperm cryopreserved using 12% GLY combined with extender-1 (83.67 ± 6.7%) or extender-2 (83.3 ± 4.7%) showed a similar motility to those cryopreserved with 15% Me2SO, but significantly lower from fresh sperm. The type of straw (0.25 or 0.50 mL) did not show any significant difference (p > 0.05) in post-thaw sperm motility. The highest values of PMI and MMP were observed for 7-day cryopreserved sperm using extender-1 in combination with 15% Me2SO (91.0 ± 2.9% and 90.0 ± 2.0%, respectively) or 12% GLY (90.0 ± 1.3% and 90.0 ± 4.6%, respectively). These results were similar to those of fresh sperm (95.3 ± 2.1% and 92.9 ± 2.5%, respectively). PMI and MMP of 1-year cryopreserved sperm using extender-1 in combination with 15% Me2SO (90.3 ± 2.5% and 89.3 ± 2.1%, respectively) or 12% GLY (90.0 ± 4.4% and 88.7 ± 2.2%, respectively) were significantly similar (p > 0.05) to those of fresh sperm. Sperm DNA integrity did not reveal any significant difference (p > 0.05) between fresh and cryopreserved (7-day and 1-year) sperm. Based on the assessed sperm vitality indicators, a cryopreservation protocol using extender-1 in combination with 15% Me2SO or 12% GLY has potential for hatchery as well as to create a germplasm bank.
Stock enhancement is a method for replenishing depleted wild finfish populations by supplementing them with hatchery-raised fish. In Taiwan, silver sea bream (Rhabdosargus sarba) is a predominant commercial species involved in stock enhancement projects. Although management agencies conduct stock enhancement projects, there are a lot of private releases without records. Stock enhancement is performed by the private aquaculture sector without accurate genetic records, potentially leading to unintended consequences for wild populations. We analyzed the genetics of 459 wild and 701 hatchery-reared specimens from nine batches produced by various hatcheries. Wild and hatchery-reared samples could be considered two separate clades by using a set of stable and informative microsatellite markers including type I (from gene introns and 3′UTR) and type II markers (randomly picked up from genome). Type I microsatellite markers could more sensitively reflect the loss of genetic diversity more than type II markers in the domestication process. All specimens were considered native by using mtDNA COI and microsatellites. The genetic composition of the wild population is relatively simple, and the estimated low contribution rate of the hatchery stock (1.3–10.9%; 6–50/459) indicated a weak but significant genetic effect of stock enhancement. Therefore, establishing standards for the stock enhancement of silver sea bream for more effective supplementation of wild populations is imperative.
While the removal of seminal plasma is a routine practice prior to equine sperm cryopreservation, this fluid contains the main source of antioxidant enzymes able to scavenge these reactive oxygen species. Therefore, stallion seminal plasma components may have an impact on ejaculate freezability. Against this background, this study was designed to investigate whether the activities of the main stallion seminal plasma antioxidant enzymes are related to sperm cryotolerance. With this purpose, 16 ejaculates were collected from 14 healthy stallions, and each ejaculate was split into two aliquots. The first one was used to evaluate the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GSR) in seminal plasma. The second aliquot was extended and then processed for cryopreservation. Sperm motility and viability were evaluated before and after cryopreservation, and ejaculates were classified as of good (GFE) or poor freezability (PFE) based on total motile and viable spermatozoa at post-thaw. We observed that, while the specific activities of CAT, GPX, and GSR were similar between GFE and PFE, that of SOD was significantly (p < 0.05) higher in GFE than in PFE. We can thus conclude that, in stallions, the specific activity of SOD in the seminal plasma of a given ejaculate might be related to its freezability.
The freshness of Goldlined seabream Rhabdosargus sarba stored at 0ºC for 12 days was assessed using quality index method (QIM), physicochemical, color and microbiological parameters. Results showed that the developed QIM scheme for Goldlined seabream Rhabdosargus sarba consisted of nine parameters, which gave total 34 demerit points. QI showed a linear relationship to storage time (QIM = 8.06× storage time-7.20, R² = 0.954), and the remaining storage time could be estimated with an accuracy of ± 6 days. The physicochemical, microbiological and sensory data were integrated and used to determine the rejection point. The storage time affected L* and W values. The shelf life of whole Goldlined seabream Rhabdosargus sarba stored at 0ºC remain stable for consumption until 9 days of storage.
Although a relatively high number of sperm quality biomarkers have been reported over the years in several fish species, sperm motility is nowadays considered the best biomarker for fish spermatozoa. The first scientific reports focusing on fish sperm motility date from a century ago, but the objective assessment allowed by computer-aided sperm analysis (CASA-Mot) systems was not applied to fish species until the mid-1980s. Since then, a high number of sperm kinetic parameters from more than 170 fish species have been reported in more than 700 scientific articles, covering a wide range of topics, such as sperm physiology, sperm storage, broodstock management, the phenomenon of sperm competition, ecotoxicology and understanding the life cycle of the species. The sperm kinetic parameters provided by CASA-Mot systems can serve as powerful and useful tools for aquaculture and ecological purposes, and this review provides an overview of the major research areas in which fish sperm motility assessment by a CASA-Mot system has been used successfully.
In this study we compared post-thaw quality of P. lineatus sperm frozen shortly after collection, with sperm frozen after dilution and transportation, and up to 6 h from collection. From each sperm sample (n=10 males) five aliquots were taken. One aliquot was diluted in the freezing medium (1 sperm:8 glucose:1 methyl glycol) and frozen ∼20 min after collection in the field (control), while the other four aliquots were transported to the laboratory where freezing took place 3 or 6 h after collection. From the transported aliquots, two were diluted 1:4 in glucose solution before transportation (diluted samples), while the other two were kept undiluted until freezing (undiluted samples). Thus the five treatments were: control, undiluted-3h, diluted-3h, undiluted-6h and diluted-6h. Post-thaw sperm was evaluated for membrane integrity, motility rate and velocities (curvilinear = VCL; average path = VAP; straight line = VSL). Post-thaw membrane integrity did not differ among the five treatments (48-60% intact sperm). Sperm motility rate was similar (P>0.05) between control (64%) and undiluted samples (60-62%) and higher (P<0.05) than that in diluted samples (35-45%), regardless the time after collection when freezing took place. Velocities were higher in control and in undiluted-3h samples (VCL of 254-265 μm/s, VAP of 219-244 μm/s and VSL of 134-147 μm/s) than in diluted samples or samples frozen 6 h after collection. P. lineatus sperm can be transported/shipped to the laboratory without decreasing its suitability for cryopreservation. Sperm should be kept undiluted during storage and be frozen within 3 h.
Background:
In order to improve the efficiency of bovine sperm cryopreservation process, it is important to understand how spermatozoa respond to differences in temperature as well as the ability to recover its own metabolism. The combination between flow cytometry approach and antioxidant enzymes activity allows a more sensible evaluation of sperm cell during cryopreservation. The aim of this study was to evaluate sperm attributes and antioxidant enzymes activity during different stages of cryopreservation process. Semen samples from Holstein bulls (n = 4) were separated in 3 treatments: fresh (37 °C); cooled (5 °C); and thawed. Evaluation occurred at 0 h and 2 h after incubation. Membrane integrity, mitochondrial membrane potential (MMP) and DNA damages were evaluated by flow cytometry; activities of antioxidant enzymes such as catalase, superoxide dismutase and gluthatione peroxidase were measured by spectrofotometry.
Results:
There was an increase in the percentage of sperm with DNA damage in the thawed group, compared to fresh and cooled, and for 2 hs of incubation when compared to 0 h. Considering MMP, there was an increase in the percentage of cells with medium potential in thawed group when compared to fresh and cooled groups. Opposingly, a decrease was observed in the thawed group considering high mitochondrial potential. Also in the thawed group, there was an increase on cells with damaged acrosome and membrane when compared to fresh and cooled groups. Significant correlations were found between antioxidant enzymes activity and membrane or mitochondrial parameters.
Conclusion:
Based on our results, we conclude that cryopreservation affects cellular and DNA integrity and that the critical moment is when sperm cells are exposed to freezing temperature. Also, our study indicates that intracellular antioxidant machinery (SOD and GPX enzymes) is not enough to control cryodamage.
Semen conservation methods have been extensively studied for fish species over the last 60 years. Cryopreservation techniques can be divided into two types: one with slow cooling rates like traditional freezing and the other with ultrarapid rates as used in vitrification. These protocols increase the time over which semen samples can be used for reproduction or sperm quality analysis. Marine fish possess greater resistance than freshwater species to variations in osmolality. This favours the application of these methods, which produce better results after thawing and
offer high biotechnological potential for aquaculture. In the last 15 years, sperm cryopreservation studies have been carried out in 32 marine species, but there are few analyses of the effects of freezing on the morphology and physiology of sperm cells. The object of this review was to provide recent data on semen cryopreservation in marine fish species and to suggest variables which may be applied in future research.
Prolonged sperm storage in the epididymis of Corynorhinus mexicanus bats after testicular regression has been associated with epididymal sperm maturation in the caudal region, although the precise factors linked with this phenomenon are unknown. The aim of this work is to determine the role of reactive oxygen species (ROS) and changes in antioxidant enzymatic activity occurring in the spermatozoa and epididymal fluid over time, in sperm maturation and storage in the caput, corpus and cauda of the bat epididymis. Our data showed that an increment in ROS production coincided with an increase in superoxide dismutase (SOD) activity in epididymal fluid and with a decrease in glutathione peroxidase (GPX) activity in the spermatozoa in at different time points and epididymal regions. The increase in ROS production was not associated with oxidative damage measured by lipid peroxidation. The results of the current study suggest the existence of a shift in the redox balance, which might be associated with sperm maturation and storage.
In recent years, biotechnology has had a significant impact on the aquaculture industry, particularly in the field of breeding. Molecular selection breeding has emerged as a novel approach to breeding. Reducing the cost of genetic information for individuals with desirable traits after breeding has become an important research direction. Cryopreservation technology allows bypassing time and space constraints in genetic breeding, simplifying broodstock management. This study presents a detailed cryopreservation method for black seabream sperm, evaluating extender type, glucose concentration, cryoprotectant type and concentration, sperm-dilution ratio, and cooling protocols. Sperm motility parameters were analyzed using computer-assisted sperm analysis (CASA) before and after two days of freezing. This involved using an RS solution with a glucose concentration of 15 g/L and adding a 5% final concentration of EG as the sperm cryoprotectant. After mixing the sperm and solution at a ratio of 1:2, we subjected it to 5 min fumigation at 5 cm above the liquid nitrogen surface before plunging it into the nitrogen. Sperm motility reached 85.46 ± 7.32% after two days. Various enzymatic activities showed changes over 20 days post-cryopreservation. This improved cryopreservation protocol for black seabream sperm is beneficial for genetic breeding and reproduction and provides reference for studying the cryodamage mechanisms of black seabream sperm.
The inflammatory cytokines TNF-β and IFN-γ are important mediators of the vertebrate inflammatory response and coordinators of the immune system in regard to NF-κB signalling pathways. In this study, the TNF-β and IFN-γ genes of yellowfin seabream, Acanthopagrus latus were identified, and the multiple sequence alignments, evolutionary relationships and gene expressions of the two genes were also determined. AlTNF-β contained a 762 bp open reading frame (ORF) encoding 253 amino acids, while AlIFN-γ contained a 582 bp ORF encoding 193 amino acids. An amino-acid sequence alignment analysis showed that these proteins have highly conserved transmembrane structural domains among teleosts. Moreover, AlTNF-β has a close affinity with TNF-β of yellowfin seabream while AlIFN-γ has a high evolutionary correlation with A. regius and Sparus aurata. In addition, the mRNAs of AlTNF-β and AlIFN-γ are widely expressed in various tissues. AlTNF-β is highly expressed in gill and intestinal tissues, and the mRNA levels of AlIFN-γ are higher in spleen, skin, and gill tissues than in other tissues. Under transportation density stress, the mRNA level of AlTNF-β was significantly elevated in the intestine of the high-density group, while AlTNF-β transcription in the gills did not vary significantly among the density groups. Furthermore, AlIFN-γ expression was increased in liver, intestinal, and gill tissues under high transportation density. The results of this study show that TNF-β and IFN-γ expression in yellowfin seabream is greatly affected by density stress. The density of 125 per bag for 4-5cm fry or 1200 per bag for 1-2cm fry is most suitable for the transportation of live fish. These results might provide a reference for further studies on the immunomodulatory response process and auxiliary function of immune stress of TNF and IFN genes in fish under density stress.
The standardization of procedures for the post-collection management of semen samples is a prerequisite for the development of husbandry techniques, as well as for the use of semen in laboratory research. Despite the relevance of the sea urchin Sphaerechinus granularis as a laboratory model, the basics related to sperm collection and storage have received little attention. The aim of this research was to optimize procedures for the management of S. granularis semen, focusing on the post-collection storage of samples. Specifically, the effects of post-collection storage conditions on sperm motility parameters were evaluated; motility was assessed by computer-assisted analysis (CASA-mot) upon activation (t0), and 15 min, 30 min and 60 min after dilution in freshly collected semen, and in chilled and cryopreserved samples. Motility parameters remained unchanged for up to 60 min after activation in both fresh and chilled semen. In cryopreserved samples, sperm motility upon activation was significantly lower than in fresh semen; however, no differences with respect to t0 values were recorded when thawed semen was cold- incubated for up to 60 min post activation, thus meeting standards required by germplasm cryobanks. Post-activation longevity showed by both fresh and stored S. granularis semen, as well as its resistance to cryopreservation, are positive spermatological features that will allow the running of easy management procedures, encouraging a wider use of S. granularis in hatchery practices, as well as in laboratory activities.
This study evaluates the quality and optimization of cryopreservation protocol for sperm of Rachycentron canadum, (Cobia) by screening various cryoprotectants, extenders, freezing protocols and equilibration times. Earlier studies on toxicity assay confirmed the suitability of the extender marine fish ringer and cryoprotectants such as DMSO, EG and DMA for long-term storage. Experiments were performed with milt along with extender, cryoprotectants (DMSO, DMA and DMSO + EG) at dilution ratio of 1:40 and equilibration time of 15 s with single step freezing protocol (−6 °C/min from 4 °C to −150 °C and in LN2 at −196 °C). The outcome of these experiments revealed that the sperm quality (motility, viability, functional integrity and DNA damage) was high with DMSO (5%) + EG (5%) compared to other individual and combination of cryoprotectants after 120 days storage. Maximum sperm motility of 62.8 ± 2.7% and viability of 72.4 ± 1.9% registered with DMSO (5%) + EG (5%). With reference to morphology, the post-thaw cryopreserved sperm of DMSO (5%) + EG (5%) showed intact sperm without any distortion. Under TEM also showed normal architecture of cellular organelles and plasma membrane. Metabolic enzymes activities (LDH, Asp and ALP), were high in post-thaw cryopreserved sperm with DMSO (5%) + EG (5%) compared to other cryoprotectants, but lesser than control. Similarly, antioxidant enzymes (SOD, GPx and LPO) registered high with DMSO (5%) + EG (5%), but low LPO compared to other cryoprotectants. Comet assay of post-thaw cryopreserved sperm of 120 days storage showed minimum DNA damage (5.84 ± 1.8%) with DMSO (5%) + EG (5%). Functional integrity of post-thaw cryopreserved sperm registered maximum of 68.7 ± 3.6% with DMSO (5%) + EG (5%). This protocol thus established remarkable cryoresistance of cobia sperm; imply to be amenable for long-term storage.
Galectins belong to the β-galactoside binding protein family, which have conserved carbohydrate-recognition domains (CRDs) and participate in innate and acquired immunity in animals. In this study, two galectin genes were cloned from Onychostoma macrolepis, OmGal-3 (galectin-3) and OmGal-9 (galectin-9). The open reading frames (ORFs) of OmGal-3 and OmGal-9 contain 732 and 978 base pairs, encoding 243 and 325 amino acids, respectively. OmGal-3 contains a C-terminal CRD, but OmGal-9 contains an N-terminal CRD and a C-terminal CRD. Two galectins were expressed at varying levels in all tissues examined, with the liver showing the highest expression. The relative gene expression levels of OmGal-3 and OmGal-9 following Aeromonas hydrophila infection were significantly up-regulated in the liver and spleen, and OmGal-9 had a greater increase than OmGal-3. The recombinant OmGal-3 and OmGal-9 proteins (rOmGal-3 and rOmGal-9) were authenticated and verified by SDS-PAGE and western blotting. ROmGal-3 and rOmGal-9 agglutinated all tested bacteria, including three gram-positive bacteria (Aeromonas hydrophila, Escherichia coli, and Vibrio parahaemolyticus) and three gram-negative bacteria (Streptococcus agalactiae, Staphylococcus aureus, and Bacillus cereus) in vivo without Ca²⁺. ROmGal-3 showed strong binding both to gram-positive and gram-negative bacteria and OmGal-9 had a stronger binding activity against gram-positive bacteria. Furthermore, rOmGal-3 and rOmGal-9 exhibited dose-dependent binding capability to two classic pathogens associated molecular pattern (LPS and PGN) and two sugars (d-lactose and d-galactose), and rOmGal-3 has better binding activity at lower concentrations in LPS and PGN than rOmGal-3. The integrated analyses indicate that the two galectins probably play an important role in innate immune defense by binding to bacterial cells via the CRD domain against pathogen infection.
This study uses the CASA system and enzyme activity kit to detect movement parameters and enzyme activities of fresh and cryopreserved yellowfin seabream (Acanthopagrus latus) sperm, which could provide theoretical support for the protection of the germplasm resources of yellowfin seabream and the sustainable and healthy development of its aquacultural industry. The results showed that MOT, ALH, BCF and other indices were significantly reduced after cryopreservation (P < 0.05). VCL, VSL and WOB and others indicators were not significantly different (P > 0.05). CAT, SOD, CK and other enzyme activities of fresh sperm decreased significantly after cryopreservation (P < 0.05), and there was no significant difference in the enzyme activity of ATP and SDH (P > 0.05). After the second freezing treatment, the enzyme activities of SOD, T-GSH, ATP, CK and SDH of sperm were significantly reduced (P < 0.05), and there was no significant difference in the enzyme activities of CAT, GR, GSH-Px and LDH (P > 0.05). When the cryopreservation time was extended from 5 days to 20 days, the enzyme activities of CAT, SOD, ATP and CK in sperm tended to increase first and then decrease, while the enzyme activities of GSH-Px and LDH tended to remain stable first and then decreased. This study indicates that cryopreservation has some effect on sperm motility parameters and that the activity compared with fresh sperm is slightly reduced. Cryopreservation causes damage to the antioxidant system of sperm to some extent. It also reduces the antioxidant capacity of antioxidant enzymes and energy metabolizing enzymes to generate energy.
Mitochondrial function is essential for sperm viability, not only from a sperm metabolism perspective, but also for improvement of sperm storage in liquid and frozen states. Bull sperm have notable metabolic variability with energy production for motility and subsequently for fertilizing capacity resulting from both glycolysis and oxidative phosphorylation. The objective of this study was to determine mitochondrial function of sperm using high-throughput Seahorse Analyzer technology in fresh semen and subsequent to freezing-thawing when there was incubation in media commonly used for sperm storage (relatively large glucose concentration) and female tract (relatively small glucose concentration). Additionally, there were determinations whether there were differences in values for fertility variables by regressing sire conception rate on values for mitochondrial variables when there was evaluation of semen from bulls with varying fertility. Media with larger concentrations of glucose inhibited mitochondrial function in fresh sperm, as indicated by less maximal oxygen consumption, spare respiratory capacity and coupling efficiency when compared to sperm in the media containing less glucose. Furthermore, there was greater (P < 0.05) mitochondrial function in cryopreserved-thawed compared to fresh samples with there being no effect of incubation media. These results indicate that mitochondrial damage from cryopreservation cannot be simply overcome post-thawing with glucose supplementation of bull semen incubation media. The increase in mitochondrial function is likely due to “non-productive” oxygen consumption to maintain the mitochondrial proton gradient. Furthermore, there was a negative association of mitochondrial proton leakage with sire conception rate indicating this could be a potential biomarker of bull fertility.
The development of cryopreservation techniques has led to important changes in animal reproductive biotechnology. However, these techniques are associated with cellular and molecular damage, affecting the mitochondrial function and quality of spermatozoa; moreover studies in fish are limited. In this work, the effects of cryopreservation on ultrastructure, mitochondrial function and antioxidant defences in Atlantic salmon (Salmo salar) spermatozoa were assessed, along with intracellular calcium (Ca²⁺)i, mitochondrial DNA sequence and sperm function (motility and fertilization rate). Significant ultrastructure alterations of the middle piece and mitochondria were observed in cryopreserved spermatozoa as compared to controls. Oxygen consumption and ATP were also significantly different in cryopreserved spermatozoa, and in spermatozoa incubated with electron transport chain (ETC) uncouplers and inhibitors. Mitochondrial membrane potential, motility, fertilization rate and Ca²⁺i in cryopreserved spermatozoa displayed significant reductions compared to fresh spermatozoa. Mitochondrial potential correlated significantly with motility and fertilization rate. A redox imbalance was observed in frozen spermatozoa due to increased lipid peroxidation and superoxide anion production as compared to fresh spermatozoa. Likewise, increased activity of glutathione peroxidase and total glutathione (GSH/GSSG), as well as reduced catalase activity, were observed in comparison with fresh spermatozoa. Our results contribute to a better understanding of cryodamage to mitochondrial functions in fish spermatozoa, and enabled us to establish potential quality assessment indicators. The data suggest that cryopreservation induces a reduction in overall sperm quality and functionality through disruption of the mitochondrial ultrastructure and function, leading to energy depletion and increased oxidative stress. This knowledge may also lead to the identification of a potential biotechnological tool for improving reproductive efficiency in species of commercial and biological interest.
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.
In this study, there was an examination of the effect on the characteristics of cryopreserved black-footed (Spheniscus demersus) and gentoo (Pygoscelis papua) penguin semen, of thawing at 37 and 5 °C. For two consecutive years, semen was collected and frozen during the April-June period from six gentoo penguins, and during the October-November period from 13 black-footed penguins. After thawing, sperm motility variables were examined by computer-assisted sperm analysis. Propidium iodide and SYBR-14 were used as fluorochromes for the examination of membrane integrity. For the gentoo penguins, no differences were detected in the values of frozen-thawed semen characteristics after thawing at 37 or 5 °C. For the black-footed penguins, however, thawing at 5 °C resulted in greater values (P < 0.05) for straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), and wobble (WOB) as compared with thawing at 37 °C. After thawing at 37 ºC, there were greater values with gentoo penguin sperm for percentage motile sperm, progressive motility, curvilinear velocity (VCL), VSL VAP, LIN, STR, WOB and beat-cross frequency (BCF; P < 0.05) than that for black-footed penguin sperm. After thawing at 5 ºC, there were no differences in values for any variables between the two species. In conclusion, thawing temperature affects semen characteristics in a species-specific manner. The present data strongly suggest that cryopreservation procedures should be adapted for use with each penguin species. Cryopreserved black-footed penguin semen should be thawed after cryopreservation at 5 ºC, while that of gentoo penguins can be thawed at either 5 or 37 ºC.
The aim of our study was to compare quality of fresh and cryopreserved semen of sex-reversed female rainbow trout and sex-reversed female brook trout. The effect of different final sperm concentrations in straws with a constant extender concentration and final glucose concentration on the sperm motility parameters of cryopreserved semen was tested. Furthermore, we examined the effect of post-thaw storage time on sperm motility parameters at optimal sperm and glucose concentrations. The effects of semen cryopreservation from sex-reversed female rainbow trout and sex-reversed female brook trout on sperm fertilizing ability at the sperm-to-egg ratios 500,000:1 and 1,000,000:1 after 0 and 60 min of post-thaw storage of semen were investigated. We have demonstrated that final sperm concentrations in straws as well as final glucose concentrations in extended semen influenced post-thaw sperm motility parameters in both species. The high post-thaw sperm motility was recorded for sperm concentrations within the range of 1.0–4.0 × 10⁹ spermatozoa ml⁻¹ for both species. Furthermore, the glucose concentration appeared to be very important for cryopreservation success and its effect was species-specific. The final glucose concentrations of 0.15 M and 0.19 M, for sex-reversed female rainbow trout and sex-reversed female brook trout, respectively, produced the highest results for sperm motility after cryopreservation. The post-thaw sperm motility was unaffected by 60 and 360 min after thawing for sex-reversed female rainbow trout and sex-reversed female brook trout, respectively. The fertilization rates of cryopreserved semen of sex-reversed female rainbow trout were high and did not differ between the investigated sperm:egg ratios after 0 and 60 min of post-thaw storage. However, fertilization rates of cryopreserved semen of sex-reversed females brook trout were lower and decreased after 60 min of post-thaw storage of semen at sperm: egg ratio 1,000,000:1. In conclusion, our results demonstrated differences in freezability between the semen of sex-reversed female rainbow trout and sex-reversed female brook trout. The semen of both species can be cryopreserved at the final sperm concentration of 3.0 × 10⁹ spermatozoa ml⁻¹. However, the influence of final glucose concentration on cryopreservation success, as well as the duration of post-thaw storage time and fertilization rates, are species-specific. In our opinion, standardizing the procedure of semen cryopreservation presented in this study is a prerequisite for the development of repeatable procedures and the future implementation of cryopreserved semen from sex-reversed females into hatchery practice.
Sperm was collected from cultured male fish and cryopreserved in 0.25 mL straws for the study of sperm cryopreservation. Different parameters were evaluated, including extender, dilution ratio, cryoprotectant type and concentration, equilibrium time, cooling height (in a two-step cooling protocol), and thawing temperature. The optimum result was obtained when the sperm was diluted at a 1:7 ratio in D-16 with 5% DMSO as a cryoprotectant, equilibrated for 20 min, held at 3 cm above liquid nitrogen for 10 min, and then stored in liquid nitrogen. After thawing in a water bath at 40 °C, the percentage of motile cells and fertilization rates of frozen-thawed sperm were 35.33 ± 2.52% and 39.00 ± 4.58%, respectively, while the corresponding rates for fresh sperm were 87.67 ± 3.06% and 88.67 ± 4.62%. We also used a programmed cooling protocol in which temperature was decreased from 4 °C to -80 °C by a rate of 30 °C/min, and then straws (0.25 ml) were placed above the surface of liquid nitrogen for 2 min before being stored in liquid nitrogen. This protocol provided a post-thaw activation rate of 36.67 ± 4.77%. Further parametric optimization is required to improve the quality of frozen-thawed sperm.
Ensuring the precise conditions during cryopreservation relies on constant sperm concentration in straws with constant cryoprotectant concentrations, which are essential for standardizing cryopreservation protocols. The aim of our study was to test the effect of different sperm concentrations in straws with constant extender concentration on sperm motility parameters of cryopreserved semen of brown trout (Salmo trutta m. fario), sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar). At the same time, the usefulness of different straw sizes (0.25 and 0.5 ml) for cryopreservation of the semen of these species was examined. Additionally, the effect of the final glucose concentration at a constant sperm concentration on post-thaw sperm motility was evaluated. The final sperm concentration in straws at a constant cryoprotectant concentration influenced the post-thaw sperm motility parameters of salmonids. The optimal sperm concentration with high post-thaw sperm motility was 2.0 × 10⁹ spermatozoa ml− 1 for brook trout, 3.0 × 10⁹ spermatozoa ml− 1 for brown trout and sea trout and 4.0 × 10⁹ spermatozoa ml− 1 for Atlantic salmon. The use of 0.5 ml straws led to higher or similar values of post-thaw sperm motility than the 0.25 ml straws. The final glucose concentration of 0.15 M produced the highest results of sperm motility after cryopreservation for all tested species. Our results demonstrated that the influence of sperm concentration in straws on cryopreservation success is species-specific within salmonids. On the other hand, the effect of glucose concentration did not appear to be species-specific. In our opinion, standardization of the semen cryopreservation protocol presented in this study is a prerequisite for the development of a repeatable procedure and hence the future implementation of cryopreserved semen in hatchery practice.
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.
Understanding the effects of environmental factors in sperm qualities will be helpful in the development of optimal artificial reproduction methods and contributes towards the knowledge base of better short- and long-term fish semen preservation conditions The objectives of this study were to determine properties and activities of wild-caught striped jewfish Stereolepis doederleini sperm contaminated with blood or seawater and compare them with data reported in the literature on other freshwater and marine fish species, for effective short- and long-term storage of fish semen. Overall, we observed that the sodium, chloride, glucose, total protein concentrations of normal sperm were not significantly different from blood- or seawater-contaminated sperm. The salinity and osmolality concentration of sperm contaminated with blood were lower than sperm contaminated with seawater and were not significantly different from normal sperm. In addition, the spermatozoa motility (SM) and duration of spermatozoa motility (DSM) in blood-contaminated sperm were higher than seawater-contaminated sperm and also not significantly different from normal sperm. The best condition for SM and DSM in normal sperm was dilution rate of 1:50. Sperm was immotile in distilled water, and cationic factors were shown to stimulate the initiation of spermatozoa activation. The maximum SM and DSM were observed in solution containing 0.4 M NaCl, 0.6 M KCl, 0.6 M CaCl2 and 0.4 M MgCl2. This study provides some basic and important knowledge about striped jewfish sperm sensitivity to a cationic condition. In this regard, Na+ is the major inhibitory factor of spermatozoa motility in this fish species.
Sperm cells can be damaged during the semen cryopreservation process, decreasing their fertilizing ability. Physical damage and oxidative stress may occur during the freeze-thawing process. Antioxidants such as the native antioxidant melatonin can potentially improve cryopreservation outcomes. In this study, we added melatonin to cryoprotectant to examine its effect on frozen-thawed human sperm. We found that adding 0.1mM melatonin to cryoprotectant significantly increased sperm viability (24.80 ± 0.46% vs. 20.97 ± 1.27%, P < 0.05) and membrane integrity (P < 0.05), and decreased intracellular reactive oxygen species and lipid peroxidation damage. Furthermore, mRNA levels of the transcription factor NF-E2-related factor-2 and its downstream genes were significantly increased. Resistance to oxidative stress was enhanced and expression of the antiapoptotic gene Bcl-2 was increased by inclusion of 0.1mM melatonin in the cryoprotectant. Moreover, 0.1mM melatonin upregulated the expression of heat shock protein 90 (HSP90), which confers resistance to stressors in frozen-thawed sperm. Results obtained upon addition of inhibitors of melatonin receptors (luzindole and 4-P-PDOT) and an HSP90 inhibitor (geldanamycin) in the cryoprotectant demonstrated that melatonin promoted HSP90 translation via the melatonin receptor MT1 and increased adenosine triphosphate levels, thus increasing the viability of thawed sperm.
The Southern Flounder Paralichthys lethostigma is a high-value species and a promising aquaculture candidate. Because sperm volume can be limited in this species (<500 µL), new sperm cryopreservation methods need to be evaluated. Vitrification is an alternative to conventional slow-rate freezing, whereby small volumes are cryopreserved at high cooling rates (>1,000°C/min). The goal of this work was to develop a standardized approach for vitrification of Southern Flounder sperm. The specific objectives were to (1) evaluate thawing methods and vitrification solutions, (2) evaluate the postthaw membrane integrity of sperm vitrified in different cryoprotectant solutions, (3) examine the relationship between membrane integrity and motility, and (4) evaluate the ability of vitrified sperm to fertilize eggs. From the vitrification solutions tested, the highest postthaw motility (28 ± 9% [mean ± SD]) and membrane integrity (11 ± 4%) was observed for 20% ethylene glycol plus 20% glycerol. There was no significant difference in postthaw motility of sperm thawed at 21°C or at 37°C. Fertilization from vitrified sperm in one trial yielded the same fertilization rate (50 ± 20%) as the fresh sperm control, while the sperm from the other two males yielded 3%. This is the first report of fertilization by vitrified sperm in a marine fish. Vitrification can be simple, fast, inexpensive, performed in the field, and, at least for small fishes, offers an alternative to conventional cryopreservation. Because of the minute volumes needed for ultrarapid cooling, vitrification is not presently suited as a production method for large fishes. Vitrification can be used to reconstitute lines from valuable culture species and biomedical models, conserve mutants for development of novel lines for ornamental aquaculture, and transport frozen sperm from the field to the repository to expand genetic resources.
Received August 23, 2016; accepted December 22, 2016
High levels of reactive oxygen species are associated with spermatozoa cryopreservation, which bring damage to functional spermatozoa. The aim of the present study was to investigate whether and how the freezing extenders supplemented with trehalose was beneficial for the survival of rabbit spermatozoa. semen was diluted with Tris-citrate-glucose extender addition of different concentrations of trehalose. Addition of 100 mM trehaose significantly improved post-thaw rabbit sperm parameters, such as motility, intact acrosome, membrane integrity and mitochondrial membrane potential. Moreover, when freezing extenders supplemented with trehalose, activities of catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of post-thaw spermatozoa were enhanced, meanwhile, reactive oxygen species (ROS) level and Malondialdehyde (MDA) content were decreased. The results suggest that freezing extenders supplemented with 100 mM trehalose resulted in less ROS level and MDA content, higher motility and mitochondrial membrane potential as well as the integrity of acrosome and plasma membrane. Supplementation of trehalose with freezing extenders is beneficial to the rabbit breeding industry.
Main features specific to sperm of fish with external fertilization are 1—the seminal fluid osmolality, and in several species, some ions prevent sperm motility in the genital track; 2—transfer from seminal fluid into sea- or freshwater triggers full motility due to osmotic and/or ionic signal; 3—immediately after activation, fish sperm swim with very high efficiency; 4—the motility period is limited to a very short duration for freshwater sperm and not for much longer in case of marine fish spermatozoa; 5—most motility parameters are decreasing and wave shape is changing during the motility period; 6—the regulation of axonemal motility by ionic concentration, as well as by ATP concentration and other substances, can be observed in vitro by using membrane-deprived spermatozoa; 7—the chemical energy available in fish spermatozoa is rapidly exhausted during the swimming period; 8—the membrane potential of the spermatozoon represents an important aspect of the cell electro-chemical energy homeostasis; 9—the ultimate task for sperm, before fertilization, is to meet an egg, and this task is facilitated by chemotaxis.
The objective was to examine if there are relationships between alterations in sperm viability, ROS synthesis and DNA integrity induced by cryopreservation of bovine sperm. Four ejaculates were collected from each of six bulls. Each ejaculate was diluted and divided into two aliquots; one was incubated for 24 h at 37 °C, and the other frozen, thawed and incubated for 24 h at 37 °C. Analyses of quality of sperm were performed after 0, 3, 6, 12, and 24 h of incubation. Progressive motility was determined with computer assisted sperm analysis. Percentages of plasma membrane- and acrosome-intact sperm (%PMAI), sperm with a high mitochondrial membrane potential (%HMMP), sperm showing a high degree of DNA fragmentation (%DFI), and their ROS content were assessed with dichlorofluorescein-diacetate (DCFH), dihydrorhodamine (DHR), diaminofluorescein diacetate (DAF-2 DA), and mitochondrial superoxide indicator (MITOSOX) using flow cytometry. While all other sperm parameters showed alterations (P < 0.05) during the 24h incubation time, %DFI stayed constant (P > 0.05, 0.91±0.23) in non-frozen sperm. Cryopreservation induced changes of all sperm parameters (P < 0.05). In contrast to all other sperm parameters, DFCH-fluoroescence indicating the synthesis of H2O2 showed a similar exponential rise (P < 0.05) like the %DFI values in frozen sperm. In conclusion, changes of DNA integrity in frozen sperm seem to be related to synthesis of H2O2, but not to sperm viability and synthesis of other reactives oxygen species.
Abstract The complete mitochondrial genome of the Rhabdosargus sarba was presented in our study. The mitochondrial genome is 16,644 bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The gene order and composition of R. sarba mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand are 27.01% of A, 17.96% of C, 26.02% of T and 29.01% of G. With the exception of the NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand.
The grouper, Epinephelus lanceolatus, is a vulnerable species of high economic value. An effective protocol was developed for the cryopreservation of E. lanceolatus by comparing different extenders produced by mixing various cryoprotectants (dimethyl sulfoxide, methanol and glycerol) and diluents (MPRS, TS-2, TS-19, Cortland and Hank's). Using computer-assisted sperm analysis (CASA) and morphological analysis, the sperm motility and fertilization rates from post-thaw sperm were comparable to untreated controls. The results revealed that MPRS (containing 12% DMSO) or TS-19 (containing 12% DMSO), were the optimum extenders for protecting the sperm from cryo-damage in liquid nitrogen. The post-thaw sperm maintained high motility (90.61 ± 3.03%) and a fertilization rate (92.27 ± 2.43%) similar (P > 0.05) to fresh sperm (94.34 ± 4% and 94.10 ± 1.87%). This study is the first to report on the successful sperm cryopreservation of E. lanceolatus and provides an important tool for repopulating this species through aquaculture.
Computer-assisted sperm analysis (CASA) systems have evolved over approximately 40 years, through advances in devices to capture the image from a microscope, huge increases in computational power concurrent with amazing reduction in size of computers, new computer languages, and updated/expanded software algorithms. Remarkably, basic concepts for identifying sperm and their motion patterns are little changed. Older and slower systems remain in use. Most major spermatology laboratories and semen processing facilities have a CASA system, but the extent of reliance thereon ranges widely. This review describes capabilities and limitations of present CASA technology used with boar, bull, and stallion sperm, followed by possible future developments. Each marketed system is different. Modern CASA systems can automatically view multiple fields in a shallow specimen chamber to capture strobe-like images of 500 to >2000 sperm, at 50 or 60 frames per second, in clear or complex extenders, and in <2 minutes, store information for ≥30 frames and provide summary data for each spermatozoon and the population. A few systems evaluate sperm morphology concurrent with motion. CASA cannot accurately predict ‘fertility’ that will be obtained with a semen sample or subject. However, when carefully validated, current CASA systems provide information important for quality assurance of semen planned for marketing, and for the understanding of the diversity of sperm responses to changes in the microenvironment in research. The four take-home messages from this review are: (1) animal species, extender or medium, specimen chamber, intensity of illumination, imaging hardware and software, instrument settings, technician, etc., all affect accuracy and precision of output values; (2) semen production facilities probably do not need a substantially different CASA system whereas biology laboratories would benefit from systems capable of imaging and tracking sperm in deep chambers for a flexible period of time; (3) software should enable grouping of individual sperm based on one or more attributes so outputs reflect subpopulations or clusters of similar sperm with unique properties; means or medians for the total population are insufficient; and (4) a field-use, portable CASA system for measuring one motion and two or three morphology attributes of individual sperm is needed for field theriogenologists or andrologists working with human sperm outside urban centers; appropriate hardware to capture images and process data apparently are available.
Abstract The aim of this study was to optimize the cryopreservation protocols for the sperm of red seabream, Pagrus major. The 2-mL cryovials and programmable freezer were employed for cryopreservation. Six extenders, six cryoprotectants in various concentrations ranging from 6 to 20% (v/v), four cooling rates, and three thawing temperatures were evaluated by postthaw sperm motility and fertility. The ratio of sperm to egg for postthaw sperm fertilization trials was experimentally standardized and was optimal at 500:1. The best motility of postthaw sperm (79.4 ± 4.7% to 88.6 ± 8.0%), fertilization rates (89.6 ± 2.9 to 95.6 ± 1.9%), and hatching rates (85.3 ± 5.1% to 91.4 ± 4.3%) were achieved when Cortland extender, dimethyl sulfoxide (15, 18, and 20%) or ethylene glycol (9, 12%) as cryoprotectants, 20 C/min as the cooling rate, and 40 C as the thawing temperature were employed. Moreover, the results on embryonic development were not significantly different between cryopreserved sperm and fresh sperm during incubation process. In conclusion, these methods of cryopreservation of red seabream sperm are suitable for routine aquaculture application and preservation of genetic resources.
The impact of successful cryopreservation of spermatozoa can be found in many fields, including agriculture, laboratory animal medicine, and human assisted reproduction, providing a cost-effective and efficient method to preserve genetic material for decades. The success of any cryobiologic protocol depends critically on understanding the fundamentals that underlie the process. In this review, we summarize the biophysical fundamentals critical to much of the research in sperm cryobiology, provide a synopsis of the development of sperm cryobiology as a discipline, and present the current state and directions for future research in sperm cryobiology in the three major areas outlined above-agriculture, laboratory animal medicine, and human clinical assisted reproduction. There is much room for new research, both empiric and fundamental, in all areas, including refinement of mathematical models, optimization of cryoprotective agent addition and removal procedures for spermatozoa from many species, development of effective, efficient, and facile cryopreservation protocols and freezing containers for agricultural sperm cryopreservation, and tailoring cryopreservation protocols for individual human samples.
This study examined the effects of storage time and cryoprotectant concentrations on the post-thaw sperm of red seabream, Pagrus major. Sperm treated with 12%, 15%, 18% and 21% DMSO were cryopreserved for 10, 30, 60 and 360 days, and fertilization and hatching rates were analysed. For all groups, there were no differences in the fertilization rates and hatching rates between sperm cryopreserved for <60 days and fresh sperm (98.8±0.8%, 96.4±1.3%). However, for sperm cryopreserved for 360 days, both fertilization rates (88.6±3.0% to 7.0±1.9%) and hatching rates (79.4±7.2% to 3.3±0.8%) decreased drastically. Furthermore, the cryoprotectant concentrations affected sperm quality significantly (P<0.05). When cryopreserved for 360 days, sperm treated with 15% DMSO obtained the best results compared with other concentrations. We suggest that 15% DMSO may be an effective cryoprotectant for long-term sperm cryopreservation of red seabream.
Experiments were carried out on the cryopreservation of common carp (Cyprinus carpio L.) sperm. Optimal conditions for fertilization including suitable medium and sperm:egg ratio were determined. Sperm was diluted in modified Kurokura's ‘Extender 2’containing DMSO as cyroprotectant in 10% final concentration. The dilution rate was 1:9 (sperm:diluent). Sperm was diluted and equilibrated (10 min) at 2°C. Sperm was then frozen in plastic straws (0.5 ml) at the following rate: 0°C–4°C: 4°C min−1; −4°C–80°C: 11°C min−1; from −80°C they were plunged directly into liquid nitrogen (− 196°C). Frozen samples were thawed in a water bath at 40°C. Fertilization rates achieved were much higher in water than in other solutions. Optimal ratios of frozen sperm:egg:water (1:20:20 in volume) and optimal number of frozen spermatozoa:egg (105 spz: 1 egg) were determined. In such conditions, a strong positive correlation (c =+0.846) was found between the post-thaw motility and the fertility of frozen sperm securing high fertilization (99.6%, percent of control). No significant difference was found between fertilization and hatching rates achieved using frozen-thawed common carp sperm.
The Brazilian flounder, Paralichthys orbignyanus, is being considered for aquaculture due to its high demand and market price. Reproduction and larviculture studies have demonstrated the feasibility of massive fingerling production, and techniques that prolong life and increase gamete viability can assist in the culture development of this species. The aim of this study was to evaluate the efficiency of two different cryosolutions for cryopreservation of Brazilian flounder semen in order to improve broodstock management and consequently augment the potential for its culture. Two different cryosolutions were tested: a) glycerol–saline: glycerol solution (12% or 1.65 M) along a saline-based diluent (423 mM NaCl, 9 mM KCl, 9.25 mM CaCl2.2H2O, 22.92 mM MgCl2.6H2O, 25.5 mM MgSO4.7H2O and 2.15 mM NaHCO3; pH 8.2; osmolality 900 mOsmol/kg); and b) DMSO–sucrose: DMSO solution (10% or 1.40 M) along a sucrose-based diluent (110 mM Sucrose, 100 mM KHCO3 and 10 mM Tris-Cl; pH 8.2; osmolality 335 mOsmol/kg). Cryopreservation was made without equilibration time. First, 250 μl-straws were placed 6 cm above the surface of liquid nitrogen for 10 min, then they were maintained for 5 min on the surface of liquid nitrogen (1 cm) before being plunged into liquid nitrogen. The quality of cryopreserved sperm was assessed through the percentage of sperm motility and viability, fertilization capacity, hatching and larval viability. Motility was estimated with an arbitrary scale, ranging from 0 to 5. Spermatozoa viability was determined using a LIVE/DEAD® sperm viability kit. Motility of fresh sperm (3.5 ± 0.2) was similar to frozen/thawed sperm with DMSO-sucrose (2.5 ± 0.3) (P > 0.05). On the other hand, the motility of frozen/thawed sperm with glycerol-saline (1.3 ± 0.4) was lower than the other two treatments (P < 0.05). No difference was found in the percentage of live spermatozoa post-thawed between DMSO–sucrose and glycerol–saline solutions (P < 0.05). However, fresh sperm had a higher percentage of live spermatozoa than post-thawed sperm with glycerol-saline (P < 0.05). Sperm motility was positively correlated with the percentage of live spermatozoa (Adjusted R2 = 0.61, n = 13). No difference was found for fertilization and hatching rates and larvae viability among the three treatments (P > 0.05). This is the first report on successful cryopreservation of Brazilian flounder sperm. This procedure should improve broodstock management techniques for this species and consequently augment the potential for its culture.
Advances in research on cryopreservation of gametes and embryos of aquatic organisms are modest compared with work done in terrestrial animals and plants. While sperm has been successfully cryopreserved in a number of cultured finfish and shellfish species, utilization at the farm level is still very limited. Modest success has been achieved in the cryopreservation of shellfish embryos and early larvae. On the other hand, cryopreservation of finfish ova and embryos has not been successful so far. Unlike shellfish eggs and embryos, finfish ova and embryos are large, contain a large amount of yolk and are covered with a relatively thick chorion. Uniformity in the penetration of conventional cryoprotectants and in cooling during the freezing process has not been attained in these large and dense specimens.Just as cryopreservation has many practical applications in breeding terrestrial plants and animals, cryopreservation also offers the same potential in the artificial propagation of many aquatic animals. Furthermore, cryopreservation has a significant role in such concerns as aquatic biodiversity, eco-toxicology, and environmental conservation.This paper reviews the recent advances in finfish and shellfish gamete and embryo cryopreservation. Work done in the authors' country is discussed separately from those in the other countries. Future cryopreservation research in aquatic animals is directed towards overcoming the problems posed by finfish ova and embryos using very recent technical innovations. The wide application of cryopreservation in fish farming and in the preservation of aquatic animals in general is encouraged.
The major objective was to investigate plasma steroid profiles after stimulation with LHRH-A or HCG during the bisexual, mature male and female phases in black porgy, Acanthopagrus schlegeli, a marine protandrous hermaphrodite. The responses of milt volume and oocyte diameters were also studied. Two-year-old black porgy during the non-spawning (bisexual stage) and spawning season (male phase), and wild-captured mature females, were injected with a superactive analogue of mammalian luteinizing hormone releasing hormone (LHRH-A) and human chorionic gonadotropin (HCG). Plasma samples were taken from fish before and after injection. Milt volume and oocyte diameters were also measured in mature males and females. Plasma testosterone in the bisexual and mature male phase increased significantly after treatment with LHRH-A or HCG. Plasma estradiol-17β and testosterone also increased in mature females after treatment. Peak levels of plasma testosterone occurred earlier in the mature male phase than in the bisexual stage. Spermiation and oocyte maturation were also stimulated during the spawning season after injection of LHRH-A and HCG. Peak levels of plasma progesterone and 17α-hydroxyprogesterone were not found after stimulation.
Successful sperm cryopreservation techniques have been developed for Eurasian sturgeon species; however, there is little information available on these techniques for North-American species. In this study, two sets of sperm cryopreservation experiments were carried out on the endangered shortnose sturgeon (Acipenser brevirostrum). In the first set, the cryoprotectants methanol (MeOH) and dimethyl sulfoxide (DMSO) were investigated using three concentrations (5%, 10% and 15%). The highest post-thaw motility was found using 5% DMSO (26±13%) while the use of 5% MeOH resulted in the highest rates for fertilization at the 4-cell stage (40±15%), neurulation (38±13%) and hatching (32±12%). In the second set, the Original Tsvetkova's extender (OT), Modified Tsvetkova's extender (MT) and modified Hanks' balanced salt solution (mHBSS) were investigated in combination with three MeOH concentrations. The highest post-thaw motility (18±10%), fertilization (18±11%) and hatching rates (17±12%) were observed with MT extender used in combination with 5% MeOH. In another set of experiments, the effects of two extenders (MT and mHBSS) and two concentrations of MeOH were investigated for sperm cryopreservation of pallid sturgeon (Scaphyrinchus albus). The highest post-thaw motility (70±10%) was observed using MT and 10% MeOH while MT and 5% MeOH yielded the highest rates of fertilization (88±6%) and hatching (73±14%). In general we conclude that although hyperosmotic conditions of extenders and cryoprotectants result in higher post-thaw motility, they seem to reduce the fertilizing ability of the sperm.
In the present study, we examined methods for the cryopreservation of Epinephelus septemfasciatus spermatozoa. The percent motility, average path velocity, and linearity of movement (LIN) of fresh and corresponding post-thaw sperm were evaluated. Sperm motility was investigated using computer-assisted sperm analysis. Five percent dimethyl sulphoxide (Me₂SO) with 95% fetal bovine serum (FBS) was the most successful cryoprotectant diluent with a comparative post-thaw motility of 77.6±8.5%; 5% dimethyl formamide was also effective. Fetal bovine serum was significantly better as an extender when compared with artificial seminal plasma, glucose, and trehalose solution. Sperm tolerated a wide range of cooling rates (from 27.1 to 94.3 °C min⁻¹); however, the post-thaw motility of sperm cooled to -30 °C was significantly lower than that of other cooled temperatures (-40 to -70 °C). The velocity of post-thaw sperm was significantly lower than that of fresh sperm, although LIN remained the same. For effective cryopreservation of seven-band grouper sperm, samples should be diluted in 5% Me₂SO with 95% FBS and cooled to at least -40 °C before immersion in liquid nitrogen.