Methods in Reproductive Aquaculture: Marine and Freshwater Species (Marine Biology (original) (raw)
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Cryopreservation of sperm in marine fish
Aquaculture Research, 2000
Since the first work of Blaxter in 1953, fish sperm cryopreservation has been attempted on about 30 marine species. The present paper reviews the techniques used and the results published in these species. Particular attention is paid to the handling procedure of sperm before freezing, the problems of semen ageing and semen contamination with urine. The quality of frozen-thawed semen was evaluated using previously standardized biotests, such as a two-step motility activation technique adapted for the different species and fertilization assays using a discriminating insemination technique. Most extenders used in marine fish are saline or sugar solutions. From the investigated cryoprotectants, dimethyl sulphoxide (DMSO) generally leads to the best results. Cooling rates range from 8 °C to 99 °C min-1; the thawing rate is generally high. Compared with freshwater species, a high percentage of spermatozoa survives cryopreservation. Therefore, and because of the simplicity of the techniques, the cryopreservation of marine fish sperm is suited for application in aquaculture.
IOP conference series, 2020
Tor soro, an endemic fish from North Sumatera, has a high economic value. Environmental damage causes a decreasing of Tor soro populations and leads to the extinction of these fishes in their natural habitat. Cryopreservation is an efficient strategy that can be used to minimize the problem. The success of this approach depends on how effective it is. This study to evaluate the fertilization of Tor soro using post cryopreservation spermatozoa 48 hours. The sperms collected by stripping, followed by the dilution using soluble liquid (fish ringer methanol 10%+skim milk) 1:9 comparison. The concentrations of the skim milk that is used in 0%, 5%, 10%, 15%, 20%, and 25%. The equilibration was carried out at the 5 o C for 10 minutes before, then it kept frozen at the temperature of-10 o C for 48 hours. The sperm thawing was carried out at the 40 o C for 1 minute and one hundred egg cells (±1 g) were fertilized using 1.5ml of post cryopreservation sperm. The ANOVA test showed there was a fertility percentage effect of the skim milk on the frozen sperm. The 10% skim milk give an optimum with the highest fertility of (p<0.05) with 91.25±2.22% and motility (p<0.05) in 10% with 82.90±1.40%.
Cryopreservation of Brazilian flounder ( Paralichthys orbignyanus) sperm
Aquaculture, 2008
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 CaCl 2 .2H 2 O, 22.92 mM MgCl 2 .6H 2 O, 25.5 mM MgSO 4 .7H 2 O and 2.15 mM NaHCO 3 ; 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 KHCO 3 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 N 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 b 0.05). No difference was found in the percentage of live spermatozoa post-thawed between DMSO-sucrose and glycerol-saline solutions (P b 0.05). However, fresh sperm had a higher percentage of live spermatozoa than post-thawed sperm with glycerol-saline (P b 0.05). Sperm motility was positively correlated with the percentage of live spermatozoa (Adjusted R 2 = 0.61, n = 13). No difference was found for fertilization and hatching rates and larvae viability among the three treatments (P N 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.
Sperm cryopreservation in fish and shellfish
PubMed, 2007
Initial success in sperm cryopreservation came at about the same time for aquatic species and livestock. However, in the 50-plus years since then cryopreserved sperm of livestock has grown into a billion-dollar global industry, while despite work in some 200 species with well over 200 published reports, cryopreservation of aquatic species sperm remains essentially a research activity with little commercial application. Most research has focused on large-bodied culture and sport fishes, such as salmonids, carps, and catfishes, and mollusks such as commercially important oyster and abalone species. However, only a handful of studies have addressed sperm cryopreservation in small fishes, such as zebrafish, and in endangered species. Overall, this work has yielded techniques that are being applied with varying levels of success around the world. Barriers to expanded application include a diverse and widely distributed literature base, technical problems, small sperm volumes, variable results, a general lack of access to the technology, and most importantly, the lack of standardization in practices and reporting. The benefits of cryopreservation include at least five levels of improvements for existing industries and for creation of new industries. First, cryopreservation can be used to improve existing hatchery operations by providing sperm on demand and simplifying the timing of induced spawning. Second, frozen sperm can enhance efficient use of facilities and create new opportunities in the hatchery by eliminating the need to maintain live males, potentially freeing resources for use with females and larvae. Third, valuable genetic lineages such as endangered species, research models, or improved farmed strains can be protected by storage of frozen sperm. Fourth, cryopreservation opens the door for rapid genetic improvement. Frozen sperm can be used in breeding programs to create improved lines and shape the genetic resources available for aquaculture. Finally, cryopreserved sperm of aquatic species will at some point become an entirely new industry itself. A successful industry will require integrated practices for sample collection, refrigerated storage, freezing, thawing, rules for use and disposal, transfer agreements, and database development. Indeed the development of this new industry is currently constrained by factors including the technical requirements for scaling-up to commercial operations during the transition from research, and the absence of uniform quality control practices, industry standards, marketing and price structures, and appropriate biosecurity safeguards.
Cryopreservation of Sperm in Red Tilapia (Oreochromis niloticus
Catrina: The International Journal Of Environmental Sciences
Cryopreservation of fish sperm is a valuable method for restoration of endangered species as well as a technique for manipulation of reproduction for genetic improvement in fish. The objective of this study was to determine the effect of equilibration time, vapour temperature and exposure time on postthawed sperm motility characteristics in red tilapia. Semen was collected from matured male, diluted using TCAYE extender in French straws (0.25 ml) and stored in liquid nitrogen tank. This research involved a 3 x 4 x 4 factorial experiment consisting of 3 equilibration times (30, 45 or 60 minutes), 4 vapour temperatures (-70,-80,-90 or-100°C) and 4 exposure times (5, 7, 9 or 10 minutes). Sperm movement and velocity distribution after frozen-thawed were evaluated using Automated Semen Analyzer (IVOS, Hamilton-Thorne). The highest percent motility was obtained significantly (P ≤0.05) when red tilapia fish sperm were equilibrated for 60 minutes (63.2±1.9%), vapourized at-80°C (61.2±2.1%) and exposed for 10 minutes (57.6±2.0%). The results from this study on red tilapia fish sperm diluted with TCAYE extender suggested that the optimal percent motilities of sperm could be obtained from combination of 60 minutes equilibration time,-80°C of vapour temperature and 10 minutes of exposure time.
Archimer Cryopreservation of sperm in marine fish
Since the first work of Blaxter in 1953, fish sperm cryopreservation has been attempted on about 30 marine species. The present paper reviews the techniques used and the results published in these species. Particular attention is paid to the handling procedure of sperm before freezing, the problems of semen ageing and semen contamination with urine. The quality of frozen-thawed semen was evaluated using previously standardized biotests, such as a two-step motility activation technique adapted for the different species and fertilization assays using a discriminating insemination technique. Most extenders used in marine fish are saline or sugar solutions. From the investigated cryoprotectants, dimethyl sulphoxide (DMSO) generally leads to the best results. Cooling rates range from 8 °C to 99 °C min-1; the thawing rate is generally high. Compared with freshwater species, a high percentage of spermatozoa survives cryopreservation. Therefore, and because of the simplicity of the techniques, the cryopreservation of marine fish sperm is suited for application in aquaculture.
Cryopreservation of fish sperm: applications and perspectives
Journal of Applied Ichthyology, 2010
Cryopreservation is of interest not only for fish farming but also for the conservation and genetic improvement of resources. This technique has been well established in some freshwater fish species mainly, salmonid, sturgeons and carps, however, only in the last decade research was focused in marine fish species. The benefits of sperm cryopreservation include: (i) synchronization of gamete availability of both sexes, (ii) sperm economy; (iii) simplification of broodstock management, (iv) transport of gametes from different fish farms, and (v) germplasm storage for genetic selection programs or conservation of species. These issues would certainly benefit the aquaculture industry. The tremendous impact that biotechnology is having in aquaculture has been particularly obvious in recent years. Several species are being used as research models not only for aquaculture development applications but also for medical research. Sperm cryopreservation can give an important contribution in the germ storage of all transgenic lines. However, in all applications in fish sperm, cryopreservation needs to overcome a lack in standardization of methodologies and procedures, a correct assay of seminal quality and the development of tools to characterize cryoinjury. Many efforts have recently been made in the study of DNA using different approaches such as the comet assay (single cell gel electrophoresis), TUNEL (terminal deoxynucleotidyl transferase-nick-end-labelling), SCSA (sperm chromatin structure assay) and the analysis of specific DNA sequences using RT-PCR, since DNA damage may impair fertility or embryo development. Cryopreservation of gametes would certainly benefit from a higher concern on male improvement, basically through nutrition or selection of resistant stocks (e.g. stress resistant individuals or highly adapted to captivity) producing gametes of higher quality. There is a huge window of opportunities for improve the resistance of cells to cryopreservation through diet supplementation of certain compounds such as amino acids (taurine and hypotaurine), vitamins (Vit. E and C) and lipids or through a direct supplementation of the extender media. An equilibrium of those compounds will improve spermatozoa and seminal plasma composition protecting cells against oxidative stress (lipid peroxidation, protein oxidation, DNA fragmentation, enzyme protection) that is gaining each day more importance in cryodamage research. Table 1 Extender composition, cryoprotectant concentration and semen: extender dilution ratio used for cryopreserve sperm from several fish species Species Extender Extender composition [Cryoprotectant] extender Dilution ratio References Rainbow trout # 6 Erdahl and Graham 0.70 mM M CaCl 2 .2H 2 O, 1.08 mM M MgCl 2 .6H 2 O, 1.49 mM M Na 2 HPO 4 , 34.30 mM M KCl, 99.95 mM M NaCl, 0.52 mM M citric acid, 55.51 mM M glucose, 2.26 mM M KOH, 6.50 mM M bicine, 1% BSA 7% DMSO 1 : 3 Lahnsteiner et al. (1996); Cabrita et al. (2001b) Brown trout # 6 Erdahl and Graham 0.70 mM M CaCl 2 .2H 2 O, 1.08 mM M MgCl 2 .6H 2 O, 1.49 mM M Na 2 HPO 4 , 34.30 mM M KCl, 99.95 mM M NaCl, 0.52 mM M citric acid, 55.51 mM M glucose, 2.26 mM M KOH, 6.50 mM M Cryopreservation of fish sperm 625
Theriogenology 78 (2012) 361–368
The objective was to develop a suitable freezing method to cryopreserve Brycon opalinus (Characiformes) sperm. Extenders (NaCl and glucose at 325 and 365 mOsm/kg), cryoprotectants (dimethyl sulfoxide ϭ dimethyl sulfoxide (DMSO) and methyl glycol ϭ methyl glycol (MG)), equilibration times (15 and 30 min), thawing temperatures (30 and 60°C), and straw sizes (0.5 and 4.0 mL) were tested. Sperm were frozen in a liquid nitrogen vapor vessel at Ϫ170°C and subsequently stored in liquid nitrogen. Post-thaw sperm quality was always evaluated in terms of motility (expressed as percentage of motile sperm), duration of motility and vitality (eosin-nigrosin staining, expressed as percentage of intact sperm). The best freezing method was also tested for fertility and hatching (expressed as the percentage of fertilized eggs). Post-thaw sperm quality was highest when sperm were cryopreserved in Glucose 365 mOsm/kg and MG, after a 30-min equilibration and thawed at 60°C for 8 s, of regardless straw size: 74 Ϯ 7% motile sperm, 47 Ϯ 4 s of motility duration, 69 Ϯ 3% intact sperm, 64 Ϯ 4% fertilization and 63 Ϯ 3% hatching. The freezing method developed in the present study was efficient and can be used to maximize larvae production for both aquaculture purposes and for conservational programs, since B. opalinus is a threatened species.