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The fate of this planet is governed by the demands and activities of its human population,
and from these demands and activities the twin themes of the present volume emerge. We have
sought to address the agricultural and conservation aspects of cryopreservation in aquatic species
-- fields that are still very much in development. This book is designed as a comprehensive
single-volume compendium of information on cryopreservation in aquatic species. It may be
used as a textbook, general reference, or research guide. Our goal was to provide a broad
overview of the principles, procedures, and perspectives necessary for development and
application of the technology of cryopreservation. We have chosen to follow the guiding
principle of the first edition to present not only what we know now, but also what we do not
know. Thus this edition is not intended to be a final or complete work, and indeed future
volumes could follow with more detailed information on genetic improvement for aquaculture
and protection of natural resources, and greater emphasis on programmatic development and
commercialization.
In preparing this volume, we wished to facilitate interaction among the groups needed for
beneficial and effective use of cryopreservation in aquatic species. We hope that it will act as a
catalyst for continued growth of an international cryopreservation community in aquatic species,
and that it will serve a useful role in education and research, and most importantly, in the
development of lines of communication among the different groups working in this field. These
were the hopes for the first edition (its success in meeting these aspirations can be assessed by
other) and remain as our primary goals for this edition.
Crayfish are among the few invertebrate species reported to possess peroxidase activity. The enzyme is found within the hepatopancreas, the principal digestive and absorptive organ of the crustacean body. Cambarus robustus, a species found in abundance in the streams of western New York, was used in this study. Homogenates of 18 hepatopancreases were assayed for peroxidase activity using guaiacol as the substrate. Although present in all organs, peroxidase activity displayed a greater than 50-fold difference between the two extremes (0.05-2.72 units/mg protein). Histochemical examination using diaminobenzidine revealed peroxidase activity within a line of cells extending along the distal two-thirds of the lengths of all hepatopancreatic tubules. The cells function to synthesize the enzyme, sequester it within vacuoles of increasing size, and eventually secrete it into the tubule lumen. Since the tubule is constantly renewed by distal mitotic activity and concomitant proximal exfoliation, this histochemical technique permits not only the examination of the ontogeny of this peroxidase-positive cell line, but also offers additional insight into the mechanism of hepatopancreatic tubule renewal.
The LD50 for a 7-day period following intraperitoneal injection of apomorphine HCL was calculated to be 158 mg/kg in rainbow trout. Intraperitoneal injection of apomorphine at doses of 60 mg/kg or greater caused vomiting of plastis balls which had been placed in the stomachs of rainbow trout. Apomorphine-induced effects included vomiting, vomiting behavior, toxicity, increased respiration, impaired motor control and equilibrium, and increased aggression. The vomiting control mechanism of trout may be similar to that described in mammals.
Nuclear DNA mass in cells from a reference species can be used to obtain high-resolution estimates of DNA mass from a target species. In our study of DNA mass in cells from 45 selected species, representing each of the major vertebrate classes, we have obtained values of from 1.5 to 110.0 pg of DNA. Because values in or near this range would be expected in the study of nuclear DNA mass in vertebrates and other organisms, the species in this report can provide a useful catalogue of references for comparative studies of DNA.
By use of flow cytometry we measured nuclear DNA content in cells from 16 stocks representing 9 species of the genus Xiphophorus. Significant differences were detected between certain stocks and species with respect to DNA content. Male-female differences were apparent in 5 of 7 stocks in which males and females were studied. Estimation of nuclear DNA content is of potential significance in connection with the genetics of sex determination and the study of taxonomic relationships.
We estimated the nuclear DNA content of unfixed erythrocytes of the grass carp Ctenopharyngodon idella by fluorescence flow cytometry using propidium iodide as a fluorochrome. Frozen erythrocytes from the domestic chicken Gallus gallus were thawed and used as an internal reference for simultaneous analysis with grass carp samples. The nuclear DNA content of erythrocytes from diploid grass carp (mean ± SD) was 2.00 ± 0.01 pg/cell, whereas triploids possessed 2.97 ± 0.03 pg/cell. Variations in the nuclear DNA content of different chickens were standardized in relation to the DNA content of fresh human leukocytes (7.00 pg/cell). The value for DNA content of the chicken erythrocytes (around 2.5 pg/cell) occupied a position intermediate between the two grass carp values, and thus provided a clear reference for discrimination between diploids and triploids.
Nuclear DNA content of erythrocytes from male and female channel catfish (Ictalurus punctatus) was determined by flow cytometry. Fourteen stocks of catfish were studied ( n = 115), including domesticated and wild fish, fish from sex-reversed populations and those produced by gynogenesis. Mean DNA content was 1.977 ± 0.010 (SD) pg DNA per cell, and mean within-stock variation was 1.28%. The stocks had an average difference of 0.19% from the species mean; no significant differences in DNA content were detected among the stocks or between males and females. The intraspecific variation among the channel catfish stocks examined is lower than that reported for other fish species. This may reflect artificial stabilization of genome size by human intervention, or alternatively, evolutionary conservatism within the genome of the channel catfish.
A rapid method to identify sex in birds is described. The method requires microliter volumes of blood, and, under appropriate conditions, results can be available within an hour of sample collection. Samples can be stored at 4 °C or -20 °C without sacrificing the ability to discriminate sex differences in DNA content. The assay will find utility in laboratory, field, and applied studies, in other classes of vertebrates, and in studies on the dynamics of genome size within and among populations.
Since the late 1970's flow cytometry has been accepted as a and accurate technique for the quantification
of nuclear DNA content (Deaven 1982). However, use of technology has typically confined to clinical
settings due to the high cost of the instrumentation and the requirement for trained personnel.
Flow cytometry recently has been applied to the study of genetic damage in wild populations. McBee
and Bickham (1988) detected DNA damage in wild rodents inhabiting a dump site contaminated with
petrochemicals, and Bickham et al. (1988) detected DNA aneuploidy in turtles found in seepage basins
contaminated with radiation. Given these few studies, there is a need to establish a data base for
the application of flow cytornetry to environmental screening.
We chose to study the western cottonmouth snake, Agkistrodon piscivorus leucostoma, which is a common
inhabitant of aquatic environments of the Southeastern United States. Accumulation of a variety
of contaminants could potentially reach high levels in predatory species given its generalized diet
which includes fish, rodents, birds, turtles, and snakes (Burkett 1966). Our objectives in this
study were: a) to examine the variation in nuclear DNA content within and among three populations
of cottonmouth snakes, and b) to survey for evidence of possible genetic damage in individual snakes.
Unambiguous identification of the sex of live birds is critical in numerous areas of avian research, including studies of alternative reproductive tactics, sexratio manipulation, and conservation biology (e.g. van Rhijn 1973, Snyder and Snyder 1989, Stamps 1990). However, identification of sex can be problematic for researchers dealing with young birds or sexually monomorphic species. Techniques currently available for identification of sex, primarily based on cytogenetics or biochemical genetics, are time-consuming, expensive, or require considerable amounts of tissue. The use of sex-specific DNA probes can overcome some of these shortcomings (Quinn et al. 1990), but this technique is time-consuming also, and probes may not be equally effective with DNA of species from divergent taxonomic groups. Other techniques such as laparotomy do not always work with nestlings or sexually immature birds, are potentially stressful, and may be inadvisable when dealing with threatened or endangered species.
The occurrence of triploid individuals within normally diploid populations has been reported in a wide variety of vertebrates, although differential viability has been noted in the affected animals. Triploid mammals generally die in utero but triploidy can occur with less dire consequences in other vertebrates such as the domestic chicken, Gallus gallus domesticus, in which an adult triploid has been described (Ohno et al., l 963). Gold and A vise ( M.76) reported spontaneous triploidy in a cyprinid fish, and within the salmonids (a group characterized by ancestral tetraploidization) adult triploids of rainbow trout, Oncorhynchus mykiss, have been reported (Cuellar and Uyeno, 1972; Thorgaard and Gall, 1979).
We measured genome size (nuclear DNA content) by fluorescence flow cytometry in 55 species of birds representing 12 different orders. Similar studies were per• formed in approximately 100 species by laboratories using absorption cytophotometry of Feulgen-stained nuclei. Although there have been apparent discrepancies in the assigned values for the species used as a reference, the values obtained in the different laboratories are generally in agreement. When the data are standardized in relation to a diploid (2C) value of 2.5 picograms (pg) of DNA for the domestic chicken (Gallus gallus domesticus), the mean for DNA content in 135 species rep• resenting 17 orders is 2.82 ± 0.33 (SD) pg with a range of 2.0-3.8 pg. Thus the genome size of birds is the most conservative of any vertebrate class and, all values considered, is smaller and more uniform in size than previous estimates would indicate. This could be explained by a previously unexplored hypothesis: that the genome of birds has evolved from a small ancestral genome that was reduced before emergence of the protoavian.
We studied DNA from the protogynous sex-changing fish Anthias squamipinnis to evaluate the recent observation that male-specific bands are identified after hybridization with Bkm, a probe originating in the W chromosome of the snake Bungarus fasciatus. Sex-specific hybridization would imply modification of DNA structure during the sexchanging process. No sex-specific Bkm fragments were identified in our study, after digestion of DNA from 15 males and 11 adult females, despite the use of 12 different restriction enzymes. However, hybridization with Bkm did produce a distinct fingerprint pattern, similar to the fingerprint patterns described for other species after hybridization with GATA (GACA) type probes. In other experiments, the pDP1007 probe, which identifies the ZFY gene in the male-determining region of the human Y chromosome, generated identical hybridization patterns in DNA from males and females of A. squamipinnis and estimation of DNA mass by flow cytometry revealed identical genome sizes.
A probe from a conserved motif of the SRY gene (sex-determining region Y), a prime candidate for the human testis-determinant. was hybridized to DNA from 23 species representing 5 vertebrate classes. Hybridization occurred in species with male or female heterogamety, in species with and without sex chromosomes and in those with temperature sex determination. Sexspecific signals were observed only in mammals. Conservation of sequences homologous with SRY through 400 million years of vertebrate evolution would indicate persistence of function. However, if SRY is the primary sex determinant in mammals, it is not clear that it has a similar function, or even one that is sex-related, in nonmammals.
We describe genome size (nuclear DNA content), and cellular and nuclear dimensions of erythrocytes in a triploid Blue-and-Yellow Macaw (Ara ararauna) and its diploid parents. The genome size of the triploid (4.23 pg) was L5 times greater than the genome size of the mother (2.80 pg) and the father (2.89 pg). The sex chromosome composition was ZZW, and was predicted correctly based on the genome size of the parents. Erythrocytes of the triploid were significantly larger than the erythrocytes of the parents. Because polyploidy has been reported only in one other family of birds (Phasianidae), the parrots and their relatives might prove to be useful in the study of avian triploidy.
Treatment of channel catfish with 0.2, 20, or 200 mg/liter of dihydrotestosterone (DHn in the water during the egg stage or during egg and sac-fry stages did not alter the expected 1: 1 sex ratio of the progeny. Feeding DHT at 200 mg/kg of feed for the first 21 days after yolk sac absorption resulted in 80% females; this proportion was increased by combining feeding with treatment of200 mg DHT/liter in the sac-fry stage (90%) or in the egg and sac-fry stage (97%). In contrast, treatment of blue catfish sac-fry with 200 mg DHT/liter, with or without the combination of feeding DHT at 200 mg/kg food, resulted in 100% female populations. Neither clomiphene citrate, an estrogen-receptor blocking agent, nor clofibrate, an inhibitor of hepatic synthesis of cholesterol, affected the sex ratio of channel catfish, and neither of these compounds altered the feminizing effect of 200 mg DHT/kg when fed in combination with DHT. The nonaromatizable androgen DHT is not as effective as many other androgens in producing paradoxical female populations of channel catfish. However, feminization of blue catfish by treatment of sac-fry indicates that this species is more susceptible to hormonal manipulation and that the period of sex determination may occur earlier in development than in channel catfish.
In amniotes, the banded krait minor (Bkm) minisatellite (GATA), the human telometric sequence (TTAGGG)7, and the Y-specific genes, ZFY and SRY, are associated with a particular sex. These sequences were studied in the channel catfish, lctalurus punctatus. However, none was sex-specific in catfish; homologs of each were present in males and females. Our data suggest that components of mammalian sex-determining systems may be widespread and shared among the vertebrates in general. Whether those components arc involved in sex determination in lower vertebrates or merely represent evolutionary precursors of sexdetermining factors in amniotes remains to be determined.
Two-parameter flow cytometry was used to measure sex differences in nuclear DNA content in the Florida Scrub Jay (Aphelocoma c. coerulescens ), a sexually monomorphic, cooperative breeder. Blood samples were obtained from 39 individuals, comprising 14 known males, 14 known females and 11 birds of unknown sex. Although significant among-session variation complicated the analysis, the standard estimate of genome size for known males (3.159 ± 0.019 pg DNA per nucleus) was significantly larger than that of known females (3.095 ± ().()19 pg). and the distributions of genome size for the two sexes did not overlap. Sex of most birds of unknown sex could thereby be assigned unambiguously. The mean value for genome size was 3.129 ± 0.036 pg (SD), and intraspecific variation was 4.63% of the mean (range 3.195-3.045 pg;). Analysis of feather pulp samples, complicated by high levels of debris and variability in the fluorescence distributions, was improved by use of electronic bitmap gating. Although flow cytometric techniques will probably not allow identification of sex in all cases, flow cytometry is nonetheless a potentially useful method for determining the sex of live birds.
A low pressure, high volume, forced-air method of incubation was evaluated for eggs of channel catfish, lctalurus punctatus, and channel catfish male x blue catfish,I. furcatus, female hybrids and was compared to the traditional paddle-type incubation method. No significant difference was observed in hatching percentage, survival, or weight of sac-fry and swim-up fry or in total weight after 21 days of feeding for 10 egg masses that were divided equally and incubated with paddles or forced air. The forced-air method was inexpensive, safe, quiet, and as effective as paddle-type aeration.
Flow cytometry is often used to detect DNA aneuploidy and mosaicism associated with malignancy or genetic damage. Yet DNA aneuploidy and mosaicism detected by flow cytometry may be more apparent than real. In contrast to the DNA mass observed for blood, we consistently found markedly different values and higher variability for DNA mass among other tissues collected from the same animal. Prepared mixtures or blood and other cells generated multiple fluorescence peaks identical to those that might be expected for aneuploid mosaicism. Moreover, analysis of tissues such as feather pulp, which contains a combination of cell types, yielded multiple fluorescence peaks that were not observed when blood alone was analyzed. Thus care should be exercised in classifying DNA values from different tissues as normal or abnormal, because the appearance of supernumerary fluorescence peaks might not always indicate the presence of abnormal cell populations.
Half-sib ictalurid families were produced when eggs from three channel catfish, lctalurus punctatus, females were fertilized with a mixture of sperm from channel catfish, blue catfish, I. furcatus, black bullhead, Ameiurus melas, and flathead catfish, Pylodictis olivaris. Sperm from all four species successfully fertilized channel catfish eggs, although individual families contained different percentages of the various crosses. The enzyme glucose phosphate isomerase distinguished the different hybrids and parental groups, and confirmed sorting of offspring based on morphology. Al 1 month of age, channel catfish male x flathead catfish female hybrids (0.36±0.07 g, mean±SD) were heavier (P < 0.05) than channel catfish male X black bullhead female hybrids (0.12 ± 0.03 g}, channel catfish.
Genome size varies considerably among vertebrates, ranging from Jess than I pg to more than 200 pg; the amount of DNA differing among individuals in a population can equal the amount in the entire structural gene complement. Recent technological advances permit evaluation of genome size variation at several levels including sub-chromosomal, chromosomal and cellular. Genome size variation may also be viewed from taxonomic levels, and across evolutionary time frames. As sources of genome size variation are identified and studied, the conundrum of the C-value paradox (lack of correlations among genome size, genomic complexity and phylogenetic status of organisms) may prove to be more apparent than real. For example, the limited and relatively constant genome size of avians may be related to the physiological constraints of flight.
We used high-resolution flow cytometry to study variation in genome size within halfsibling families of hybrid catfishes produced from parents representing three genera of the family lctaluridae Genome size of hybrid offspring was exactly intermediate to the genome sizes of parental stocks, indicating that nuclear DNA segregates as a function of haploid ONA content and is stable within intergeneric hybrids. Thus, measuring genome size could provide a useful method for identifying hybrids within natural populations of fish or other organisms. Within-group variation in genome size was less than 2.5% for all groups studied, including outbred parental stocks. These values may represent the minimal level of genome size variation detectable within populations by analysis of interphase nuclei. The variation in genome size of ictalurid catfishes is small compared to variation observed in other vertebrates, and may be due to evolutionary conservatism within the catfish genome or to the appearance in other taxa of mechanisms that generate variation in DNA mass.
We report the rare natural occurrence of triploidy in a brown bullhead Ameiurus (formerly lctalurus) nebulosus. This is to our knowledge the first report of naturally occurring triploidy within the family Ictaluridaie. The nuclei of blood cells were studied by flow cytometry, volume measurement. and computer-assisted image analysis. The tripioid bad a genome size of 3.29 pg DNA/cell compared to 2.23 pg DNA/cell (SD 0.02 pg) for 28 diploid brown bullheads. Volume measurement and image analysis were able to detect the triploid condition: however, flow cytometry was the most precise technique and had the greatest resolution.
A simple and inexpensive procedure is described for the modification of standard compact refrigerators for use as incubators at temperatures between 0°C and room temperature.
We developed methods for cryopreserving sperm of channel catfish Ictalurus punctatus and evaluated the use of cryopreserved sperm for reproduction. Five cryoprotectants were evaluated: methanol, glycerol, dimethyl sulfoxide (DMSO), sucrose, and polyvinylpyrrolidone. We measured the motility of sperm that had been stored at 4°C in three concentrations of cryoprotectants (5%, 10%, 15%) dissolved in a modified Hank's balanced salt solution. All cryoprotectants reduced motility within 6 h; 5% methanol and 5% DMSO caused the smallest reduction. After sperm were frozen at -80°C and stored for 2 d at - I 96°C, motility was highest (5-10%) in samples cryopreserved with 5% and 10% solutions of methanol. Sperm cells cryopreserved in methanol solutions (5%, 10%, and 15%) were used to fertilize channel catfish eggs from three females. Fertilization ranged from 24% to 97%, and no difference in fertilization success was found between cryopreserved sperm and untreated sperm from the same males. Growth of channel catfish produced with cryopreserved sperm was not different from the growth of siblings produced with untreated sperm. Sperm cryopreservation offers utility as a routine method for gamete storage and genetic improvement of catfish.
We evaluated two methods, boiling and microwave irradiation, for the rapid isolation of DNA from barbel, adipose fin, and blood of channel catfish lctalurus punctatus. Compared with routine DNA isolation methods, these procedures were fast (microwaving, 2-4 min; boiling, 10-18 min), simple, and inexpensive (about US$0.10/fish). Samples of DNA isolated from barbels of small (mean ± SD, 2.9 ± 1.0 g) and large (290 ± 43 g) fish were of high purity (ratios of absorbances at 260 and 280 nm, A260/A2so = 1.72-1.90) and were between 0.29 and 0.67 µg/µL in concentration. Samples of DNA isolated from barbel, adipose fin, and blood of small fish, by either method, were used successfully for analysis by polymerase chain reaction (PCR). Samples isolated by boiling of barbel and blood from large fish also proved useful for PCR analysis. These DNA isolation procedures would be useful for rapid genetic screening of channel catfish. Removal of the barbel for tissue analysis would also enable direct marking of fish, and after analysis, individuals designated for further study could be identified.
Gynogenesis is a naturally occurring phenomenon in lower vertebrates in which offspring receive two sets
of chromosomes from the female (Dawley 1989). This occurs when development is activated by sperm, but genetic
material from the male is not incorporated into the embryo. Normally, haploid offspring result that do not
survive to hatching, although in certain instances suppression of meiosis II or first mitosis can result
in diploid gynogenetic offspring (Purdom 1993). Artificially induced gynogencsis has been used in the breeding
and genetic study of several fishes, including salmonids (Chourrout 1982; Allendorf et al. 1986), tilapia
(Don and A vtalion 1988), and channel catfish (Liu et al. 1992). Gynogenesis has been applied to production
ofmonosex populations, isogenic populations, and inbred lines of fish (Ihssen et al. 1990; Tave 1993).
Gynogenesis can be induced artificially by irradiating sperm with ultraviolet (UV) radiation to inactivate
genetic material. Eggs are fertilized with the irradiated sperm and shocked by temperature or pressure change
to restore diploidy.
Irradiation is usually accomplished by exposing sperm to a UV radiation source providing specified
power per unit area (µ,WI cm2). In general, radiation dosages are estimated by trial and error and reported
as time of exposure or distance of sperm from the radiation source (Chen et al. 1986; Hussain et al. 1993).
Success of genetic inactivation of sperm is typically estimated by the production of haploid or gynogenetic
embryos (Varadaj 1993). Success of irradiation, although reported less often, is estimated by the reduction
of sperm motility (percentage of actively swimming sperm upon activation with water) (Peruzzi et al. 1993).
The purpose of this study was to establish a general relationship between UV irradiation and motility
of channel catfish sperm. The authors used a commercially available UV source, a DNA crosslinker,
equipped with a photo-diode that enabled direct monitoring of total irradiation and delivery of specified
UV exposures (µ,W/cm2 x s = Joules/cm2). The objectives were to: l)measure the effect of sample
concentration on motility of irradiated channel catfish sperm; 2) measure the effect of sample depth
on the motility of irradiated channel catfish sperm, and 3) develop a species-independent formula to
estimate exposure time or distance from the UV source when metering is not available. The motility values
for irradiated sperm observed in this study encompassed the range of values reported for artificiallyinduced
gynogenetic production of channel catfish and other fishes.
Cost estimates were developed for equipping egg incubation troughs for channel catfish (lctalurus punctatus) to operate with forced air delivered via air stones or perforated pipe rather than with traditional gear motors and shaft-mounted paddles. Capital and operating costs were compared on a per-trough basis for the three configurations. A partial budget was developed to evaluate conversion from the traditional configuration to each of the forced-air configurations. Although forced-air configurations appear to be less expensive to install and operate, cost estimates suggest that two to four operating seasons may he required to recover the cost of converting an existing motor-and-paddle configuration to forced air.
Sperm of Mekong giant catfish (Pangasius gigas; formerly Pangasianodon gigas) were cryopreserved in 5 or 9% dimethyl sulfoxide (DMSO) in bicarbonate buffer (BCB) or calcium-free Hanks' balanced salt solution (C-F HBSS) at different cooling rates (-5, -12, -22 or -120 °C•min-1) and stored in liquid nitrogen for 3-4 months. Pooled ova of three female Gunther's walking catfish (Clarias macrocephalus) were inseminated with cryopreserved semen of Mekong giant catfish and fresh semen of the walking catfish (control). There was no significant difference in fertility of sperm cryopreserved in the two extenders. Best results were obtained when sperm were cryopreserved in 9% DMSO in either extender in 5 ml cryotubes and frozen at -12 °C·min-1. The percentage of fertilization was 65-66% (actual) and 73-74% (control).
The effects of long-term feeding of cottonseed meal on growth, testis development, and sperm motility of male channel catfish lctalurus punctatus were studied. Brood-sized mane channel catfish were stocked into 0.04-ha earthen ponds in April 1992 at the rate of 120 fish per pond. The fish were fed a diet (32% protein floating catfish feed) containing either 0, 25, 37.5, or 52% cottonseed meal to satiation daily, except for winter months when fish were fed on days when the water temperature was 15 C or above. Fish were harvested in July 1994. Feed consumption was similar for fish regardless of dietary treatment. Feed conversion was higher for fish fed the two highest levels of cottonseed meal and weight gain was depressed in these groups. Testis weight, gonosomatic index, and sperm motility were not negatively affected by high levels of dietary cottonseed meal indicating that reproductive capacity was not diminished. Thus it would appear that up to 52% cottonseed meal could be used in the diets of brood-sized channel catfish unless maximum gain is important. In a practical situation, growth rates of brood-sized channel catfish are not as important as reproductive performance.
A tank system with recirculating water designed for use with disposable plastic 3-L beverage bottles is described. The system consisted of 100 culture units (2-L working volume in each), five 40-L tanks, and an upwelling biofilter. The 40-L tanks were used to increase the total volume of the system, to allow operation of the filter when bottles were not occupied, and for temporary storage of fish when treatments were pooled following screening. Each bottle received water from a common distribution manifold, and flow was adjustable from O to 1 L/min (0--30 water changes per hour). Bottles could be removed easily for replacement or to facilitate study of fish. The system was designed to allow separation of treatments for genetic studies, but would be useful for experiments requiring replication of treatments or experimental units with minimal environmental variation.
Channel catfish leucocytes cryopreserved with glycerol or dimethyl sulphoxide (DMSO) had significantly higher (P<0·05) viability and recovery rates than did cells cryopreserved with methanol. After 7 days of frozen storage, a 24 to 27% reduction of viability was observed for cells cryopreserved with glycerol; a 25 to 43% reduction for cells frozen with DMSO, and a 67 to 100% reduction for cells frozen with methanol. The concentration of cryoprotectants affected the viability of cryopreserved cells significantly (P < 0.05). The viability reduction was 36% for cells frozen with 5% of cryoprotectants, 30% for cells frozen with 10% of cryoprotectants, and 49% for cells frozen with 15% of cryoprotectants. The viability of cells frozen at the slower rate ( - 2.7° C min^-1) was significantly higher (P < 0.05) than that of cells frozen at the faster rate ( - 45° C min^-1). Best results were obtained for cells cryopreserved with 10% of glycerol or DMSO and frozen at the slower rate. The chromosomes prepared from cells cryopreserved using this procedure were identical to those prepared from fresh cells, and to those reported in the literature for channel catfish.
Cryopreservation of fish sperm has applications in preserving genetic resources from stocks of endangered fishes, replenishing fisheries, reducing the number of males needed in hatchery situations, and allowing repeated spawning of specific males. As part of a larger study on artificial breeding of sciaenid fishes, we developed procedures for collection, handling, refrigerated storage, and cryopreservation of spotted seatrout sperm. Hanks' balanced salt solution (HBSS) was used as an extender for collection and storage of sperm. Sperm motility in relation to graded concentrations of HBSS was used to determine the osmolality at which sperm were activated. Based on these findings, HBSS was prepared at 201 mOsm/kg as an extender for sperm storage. To determine if ions present in HBSS were involved in sperm activation, separate activating solutions were prepared by the addition of NaCl, CaCL2, KCl, Na2HPO4, or MgSO4 to aliquots of a stock glucose solution (185 mOsm/kg). The chemicals were added at the concentration of each found in 1-x HBSS. Only the glucose solution containing 8 g/1 NaCl (424 mOsm/kg) produced activation of sperm. We also evaluated four chemicals as cyroprotectants: methanol, glycerol, dimethyl sulfoxide (DMSO), and n,n-dimethyl acetamide. Two freezing rates were evaluated by placing samples at either of two heights within a nitrogen vapor shipping dewar. The highest post-thaw motilities were in 10% DMSO with an average retention of 60% of initial motility at the lower position in the dewar, and 37% at the upper position. A third freezing rate was produced using a computer-controlled freezer programmed for a rate of -45°C/min, yielding a retention of initial motility of 31 %. Our freezing and transport of cryopreserved sperm in shipping dewars demonstrate the utility of this procedure for field applications.
Morphological comparisons, meristic counts and measurements of the parental species (Pangasius gigas and P. hypopthalmus) and the hybrid were presented Gut contents, the gastrointestinal tract (GIT), growth and carcass compositions were also studied The live weight of the 2.5--year-old farm-raised fishes were significantly different among parental species and hybrid, with P. gigas ( ~ 8 kg) being the largest, the hybrid ( ~ 3.5kg) intermediate and P. hypopthalmus ( ~ 2 kg) the smallest P. gigas had the he combined weights of the fillets and belly flap roughly the same in percentage (52-53%) as those of the hybrid (49%), but higher than that of P. hypopthalmus (47%). There was no significant difference among the fillets of subadults in the percentages of protein and moisture. The fat content of fillets was significantly higher in P. gigas ( ~ 3%) than in the hybrid (O.6%) and P. hypopthalmus.
Refrigerated storage of sperm is useful for genetic study and artificial breeding of fishes. Due to the potential loss of donor males, storage is important in species such as channel catfish lctalurus punctatus from which sperm cannot be stripped. This study addresses short-term storage {at 4 C) of channel catfish sperm by evaluation of storage methods employed for other species and for cryopreservation of channel catfish sperm. The objectives were to evaluate: I) storage of intact testes and storage of sperm suspended in an extender solution; 2) use of various storage containers with and without supplemental oxygen; 3) use of extender solution with and without the addition of an antibiotic/antimycotic cocktail; 4) use of extender solution with and without the addition of methanol; and 5) use of extender solution with and without the addition of glucose and methanol. Sperm suspended in extender solution retained motility significantly longer (9 d) than did sperm in intact testis (2 d). Sperm stored in Zip-loc plastic bags inflated with pure oxygen retained motility significantly longer ( 12 d) than did sperm stored in Zip-loc3t. plastic bags without supplemental oxygen (7 d), or sperm stored in plastic beakers (8 d) or lest tubes (8 d) without supplemental oxygen. Sperm stored with the addition of antibiotic/antimycotic cocktail or methanol retained motility significantly longer (J0,,12 d) than did sperm stored without additives (6-»8 d). Sperm stored in extender solution without glucose retained motility significantly longer ( l 9- 21 d) than did sperm stored in extender with glucose ( 13-16 d). Motility was retained for as long as 21 d in sperm stored in extender solution with 5% methanol and without glucose. In each experiment, loss of motility was associated with bacterial growth.
Artificial spawning of channel catfish Ictalurus punctatus relies on the removal of testis and suspension of sperm in an extender solution for storage and use in fertilization. Little is known about the relationships among osmotic pressure, sperm activation, motility, and storage. Our objectives were to (I) estimate motility of channel catfish sperm diluted in solutions ranging in osmotic pressure from 8 to 295 milliosmols (mosmol)/kg, (2) identify the osmotic pressure that induces threshold activation (10% motility) and the highest pressure that induces complete activation, (3) determine the role of ionic dilution in activation by use of ion-deficient solutions, and (4) evaluate the effect of osmotic pressure on the retention of motility during storage. Motility (percentage of actively swimming sperm) wa, estimated in diluted Hanks' balanced salt solution (HBSS) and sucrose solutions over a range of osmotic pressures. The HBSS osmotic pressures of threshold and complete activation were 218 +/- 15 mosmol/kg and 132 +/- 9 mosmol/kg, respectively. We found that osmotic pressures above 220 mosmol/kg induced minimal (<10%) activation and pressures below 130 mosmol/kg induced complete activation. Within the zone of incomplete activation (220-130 mosmol/kg) a decrease of 15 mosmol/kg in osmotic pressure increased motility about011<,. We used sucrose solutions to provide an osmotic environment with minimal ionic influence for the testing of activation. Osmotic pressures for threshold (214 +/- 1 mosmol/kg) and complete (125 +/- 2 mosmol/kg) activation were consistent with those obtained with diluted HBSS, suggesting that reduction in osmotic pressure play,, a major role in the activation of channel catfish sperm. Sperm stored in 122 mosmol/kg HBSS lost 82'k of initial motility after 2.5 h. However, sperm stored in solutions with higher osmolalities retained motility significantly longer (P = 0.0017). A minimum reduction of osmotic pressure to below 130 mosmol/kg is essential to induce complete motility of channel catfish sperm for motility estimates and artificial fertilization.
Motility of sperm cells of the catfishes Pangasius gigas and Pangasius hypophthalmus was estimated over a range of osmotic pressures. The osmotic pressure of threshold activation (25% motile sperm cells in a sample) was 240 :!:: IO mOsm/kg for the sperm of P. gigas. and 221 :!: 12 mOsm/kg for P. hypophthalmus. The osmotic pressure of complete activation (highest percentage of motile cells observed in a sample) was 186 ± 9 mOsm/kg for the sperm of P. gigas, and 98 :: 21 mOsm/kg for P. hypophthalmus. These experiments were performed using dilutions of calcium-free Hanks' balanced salt solution (C-F HBSS) which is highly ionic. Similar experiments \,)ere performed using diluted glucose solutions to evaluate the activation of sperm in ion-deficient solutions. The osmotic pressure in glucose solutions of threshold activation was 248 ± 27 mOsm/kg for sperm of P. gigas, and 209 :!: 16 mOsm/kg for P. hypophthalmus ; the osmotic pressure of complete activation was 187 ± 12 mOsm/kg for sperm of P. gigas, and 98 ± 24 mOsm/kg for P. hypophthalmus. These values were not significantly different from those obtained using diluted C-F HBSS. The role of particular ions in the activation of sperm of these species is unknown. Reduction in osmotic pressure appears to play a major role in activation, with the sperm of P. gigas being more sensitive to reduction of osmotic pressure than the sperm of P. hypophthalmus.
We used plastic bags to incubate, transport, and hatch embryos for specific crosses of razorback suckers Xyrauchen texanus during two spawning seasons on the Colorado River (Nevada-Arizona). Fifty to 2,000 razorback sucker eggs were fertilized in plastic food storage bags for 26 matings in 102 trials. Experimental treatments prevented hatching in all bags, but hatching was observed when fresh milt and viable eggs were used (10-99% hatch). Observation of embryo development and treatment effects was facilitated by the transparency and portability of the bags. Water volume and exchange rates were chosen according to estimated metabolic rates, and the addition of oxygen to the bags was evaluated to increase holding times. Plastic bags were inexpensive and useful for gamete storage, controlled propagation, and transport of gametes, embryos, and fry under field conditions. These techniques are suitable for various applications in studies of reproductive biology, and they are especially suited to genetic management and research on endangered species that cannot be removed from the wild.
Various aspects of the cryopreservation of spermatozoa of channel catfish (Ictalurus punctatus) were studied in relation to spermatozoa motility. The objectives were to evaluate 1) the efficacy of 5, 10 and 15% of methanol or n,n-dimethyl acetamide (OMA) as cryoprotectants; 2) the acute toxicity of 5, 10 and 15% methanol or OMA; 3) the use of 0.5-ml vs. 0.25-ml straws; 4) the efficacy of 5, 10 and 15% of methanol in Hanks' balanced salt solution (HBSS) or HBSS without glucose, and 5) the use of HBSS with or without 5% methanol. We found that use of 5% methanol as a cryoprotectant resulted in significantly higher post-thaw motility (P = 0.0001) than did 5, 10 or 15% OMA. The use of 5% of either cryoprotectant resulted in significantly higher post-thaw motility (P = 0.0001) than did 10 or 15% of the cryoprotectants. Samples containing 10 or 15% OMA had significantly lower motility (P = 0.0001) after 30 min exposure than did samples containing 5, 10 or 15% methanol. The use of 0.25-ml straws resulted in significantly higher post-thaw motility (P = 0.0001) than that of 0.5-ml straws. No difference was found in post-thaw motility between HBSS with and without glucose as the extenders. Cryopreservation in HBSS without addition of cryoprotectant resulted in post-thaw motility values of about 1%.
We developed methods for the collection, handling, and refrigerated storage of sperm from the endangered razorback sucker Xyrauchen texanus. Sperm samples were obtained from 56 of 78 male razorback suckers collected from Lake Mohave (Colorado River) during the 1994 and 1995 spawning seasons. There were significant differences among the osmolalities of blood plasma, ovarian fluid, and seminal plasma. Maximum sperm motility was observed 3 s after dilution with river water and was maintained for 16 +/- 8 s (mean 2- SD). Motility ceased 70 +/- 32 s after the addition of water. Given the short time that sperm were motile, care should be taken to ensure complete and early mixing of gametes during artificial spawning of razorback suckers. Refrigerated sperm (4°C) retained more than 90% motility when stored at 290 milliosmols/kg in calcium-free Hanks' balanced salt solution (C-F HBSS). Sperm diluted 1: 1 with C-F HBSS had significantly higher motility compared with motility of undiluted sperm at the end of 24 h. Motility of sperm diluted with three proportions of C-F HBSS decreased at similar rates during 8 d of storage. Gamete collection and storage offers potential for enhancing management efforts for razorback suckers by allowing increased control of artificial spawning. In this regard, sperm may be collected by sampling crews and stored for days at the hatchery for production of fry.
An image analysis system based on the Macintosh personal computer and public domain software (NIH Image, version 1.57) allows capture of digitized images for d.ata analysis, Three techniques for image capture and aquisition of linear and area measurements from digitized images were tested: (1) estimation of egg size, (2) estimation of number of eggs or fry, and (3) measurement of fish length. For estimation of egg size, egg samples were taken by catheterization from two female channel catfish Ictalurus punctatus. Egg diameters measured manually (1.82 +/- 0.27 and 2.59 +/- 0.19 mm) wen· not significantly different (P = 0.08 and 0.20) from the diameters estimated with image analysis (1.92 +/- 0.21 and 2.63 +/- 0.17 mm). For egg counting, channel catfish eggs were hand-stripped into Hanks' balanced salt solution and divided into seven 15-mL aliquots. The number of eggs was determined manually and images were captured for analysis. The number of eggs determined manually (436 +/- 41) was not significantly different (P = 0.80) from the number estimated with image analysis (430 +/- 43). Also, total lengths were measured manually on 50 fingerling channel catfish and 50 fingerlings of the hybrid channel catfish X black bullhead Ameiurus melas. An image of each fish was also recorded for measurement by image analysis. Replicate sets of measurements were made by three individuals with different levels of experience with the computer system. The mean (±SD) of total lengths measured manually for the channel catfish fingerlings (242 +/- 33 mm) was not significantly different (P = 0.37) from the lengths determined by image analysis (237 +/- 33, 235 +/- 33, and 231 :+: 34 mm). The mean of total lengths measured manually for the hybrids (157 +/- 39 mm) was not significantly different (P = 0.98) from the lengths measured by image analysis ( 158 ± 40, 157 +/- 39, and 155 +/- 39 mm). The availability of public domain software and inexpensive or commonly available input devices make image analysis more accessible to researchers.
Inexpensive hatching and incubation jars have been constructed in the past from polyvinyl chloride fittings or plastic bottles. Most cost less than $50 each, but contain numerous parts. The incubation jar described in this report was easy to assemble. recyclable, and cost about US$2.50. It took about 1O min to construct and 24 h for the sealant to cure before use. Eggs of common carp Cyprinus carpio in quantities of 2,000- 80,000 were hatched in these jars by adjusting water flow. The small size (2.3-L volume) provided separation of many treatments in a confined area.
Procedures were developed for the collection, refrigerated storage and cryopreservation of black drum spermatozoa. Sperm samples were collected by removing and slicing the testis, and suspending the spermatozoa in Hanks' balanced salt solution (HBSS) at 200 mOsm/kg. Threshold activation ( I 0%) of black drum spermatozoa occurred at 370 mOsm/kg, and complete activation occurred at 580 mOsm/kg in HBSS. Sperm cells activated in artificial seawater had higher motility than those activated in HBSS at osmolalities from 350 to 500 mOsm/kg. Spermatozoa stored at 4°C in HBSS or artificial seawater at osmolalities from 202 to 290 mOsm/kg retained motility longer than did those stored at other osmolalities. Dilution rate had no effect on sperm storage time at 4°C. Four chemicals were evaluated as cryoprotectants: dimethyl sulfoxide {DMSO), n,n-dimethyl acetamide (DMA), methanol, and glycerol. Glycerol and DMA at concentrations of I 0% significantly reduced motility within 52 min. Spermatozoa were cryopreserved at 3 freezing rates (-27, -30, or -45°C/min) in a nitrogen vapor shipping dewar or a computer-controlled freezer. Spermatozoa frozen using I 0% DMSO had the highest post-thaw motility at a freezing rate of -27 or -30°C/min. Spermatozoa frozen using 5% glycerol, 5% DMSO, or I 0% DMSO had the highest post-thaw motility at a freezing rate of -45"C/min.
An in-situ polymerase chain reaction (ISPCR) procedure was applied to chromosomal localization of the gene, lg H, encoding the immunoglobulin heavy chain of channel catfish (Ictalurus punctatus). Metaphase chromosomes were prepared by a replication banding procedure and subjected to ISPCR using biotin-labeled primers. The hybridization signals were detected with an avidin-fluorescein isothiocyanate (FITC)-based method, and chromosome bands revealed by simultaneous or sequential treatment methods. Standard fluorescent in-situ hybridization (FISH) was performed on chromosome preparations to compare with the ISPCR procedure. The lg H gene was detected at the telomeric position of a chromosome with a relative length of 3.2 ± 0.2%. The lg H-bearing chromosome detected by the FISH method was identical to that found by ISPCR procedure. Visibility of chromosome bands was reduced by heat and salt treatments and could not be analyzed after thermocycling. Therefore, specific identity of the chromosome bearing the lg H gene remains unknown. Banding of fish chromosomes is difficult and poses a barrier for applying current molecular techniques to physical mapping of teleost genomes. Application of the ISPCR to chromosomal mapping is new for fish species and is only in initial stages of development for higher vertebrates.
Only limited research has addressed the effect of salinity on hatching of channel catfish lctalurus punctatus eggs, and no studies have evaluated the effect of salinity on fry development and survival. This study was undertaken to determine the effect of environmental sodium chloride (0, 1, 2, and 4 g/L NaCl) on percent hatch, yolk utilization, and survival of channel catfish fry. Experiments were conducted in recirculating systems using seven egg masses (1-2 d old). Each egg mass was divided into smaller portions which remained undissociated or were dissociated with sodium sulfite (NaSO3). Eggs were incubated until hatching. Wet and dry weights were obtained for sacfry at l and 5 d post-hatch to determine wet weight gain and dry weight loss, and fry were sampled 7 d after initiation of exogenous feeding to determine survival. Percent hatch, yolk utilization, and survival of fry hatched from undissociated eggs were greatest at 1 g/L NaCL In addition, treatment of eggs with NaSO1 significantly reduced percent hatch at all NaCl levels. Although our results indicate that addition of NaCl to hatchery water supplies can increase production of channel catfish fry, additional research is needed before this practice can be recommended on a commercial basis.
This study was designed to simulate conditions encountered routinely during refrigerated storge of channel catfish sperm. Sperm samples were stored at 4 C in non-sterile and sterile Hanks' balanced sail solution (HBSS). Non-sterile HBSS was prepared with distilled water stored for 2 wk in a plastic carboy prior to use. Observations were made on the frequency and abundance of bacteria in samples, and on changes in sperm motility and quality. Sperm samples stored in non-sterile HBSS had a complete loss of motility within 72 h. Samples maintained in sterile HBSS showed an initial decrease in motility between 48 and 72 h, and a complete loss of motility within 10 d. Quality of the sperm in each buffer decreased as motility decreased; morphologic changes and reduced motility of sperm were coincident with increased bacterial numbers. Bacteria were cultured on tryptic soy agar and Pseudomonas F agar (PFA) by spread-plating 10-µL, aliquots from each sample onto bacteriologic media and incubating for 5 d. The dominant bacteria observed were members of the genus Pseudomonas., representing 67% of the total bacteria identified. The dominant pscudornonad (Pseudomonas sp.) cultured from sperm samples stored in sterile buffer produced caseinase, lccithinasc, and was 13-hemolytic, whereas the dominant bacteria (P. putida) cultured from samples stored in the non-sterile buffers did not. Highly motile pseudomonads, present in two samples stored in sterile buffer, colonized below the surface of the PFA media at 4 C. The attributes of the bacterial contaminants that likely contributed to the decrease in sperm quality were production of extracellular enzymes, consumption of oxygen, and a high level of motility. Potential sources of degradative bacteria were commensal flora of channel catfish and the water used in preparing the storage buffer.
Hybrid chromosomal compositions of channel catfish lctalurus punctatus x black bullhead Ameiurus melas and channel catfish x flathead catfish Pylodictis olivaris were analysed by a computer-based method. The karyotype of each hybrid was highly asymmetric, and the diploid numbers and arm numbers were intermediate to the parental types. The hybrid offspring of channel catfish x black bullhead possessed a diploid number of 59 chromosomes, with an arm number estimate of 87. The hybrid offspring of the channel catfish x flathead catfish cross possessed a diploid number of 57 chromosomes, also with an arm number estimate of 87 . Nucleolus organizer regions (NORs) were located on a single pair of chromosomes with symmetric staining intensity in channel catfish and in black bullhead, and on a single pair of chromosomes with asymmetric staining intensity in flathead catfish. The channel catfish x black bullhead hybrid had two unpaired chromosomes that stained positively for NORs. The channel catfish x flathead catfish had three unpaired chromosomes that stained positively for NORs. Specific marker chromosomes were identified in each hybrid. There was no evidence of androgenesis, gynogenesis, polyploidy or aneuploidy in the hybrids. Results of this study, plus information reported previously, indicate that chromosomes of ictalurid catfishes are inherited stably in a haploid pattern with an equal contribution to the genomes of F1 hybrids, even in intergeneric crosses involving divergent numbers of parental chromosomes.
Morphological abnormality (haploid syndrome) of fry was obtained from crosses between ultraviolet-irradiated (0.3 J/cm^2) eggs of Clarias gariepinus and fresh sperm of C. macrocephalus. The abnormai fry had a short, curved tail and an enlarged cavity between the yolk sac and anterior part of the body. Chromosome counts and analysis of genome size indicated that the abnormal fry were haploid, and amplification by polymerase chain reaction of genomic DNA using primers targeting a portion of the gene encoding the im munoglobulin heavy chain confirmed inheritance of the paternal genome. These findings will aid in distinguishing diploid from haploid androgenetic offspring.
Clarias gariepinus eggs were fertilized with sperm of Pangasius hypophthalmus (sutchi). Paraffin sections were made at different times after fertilization to determine the timing of the first mitotic division. The UV- irradiated (240 mJ/cm^2) eggs were also used to produce androgenetic offspring. To induce diploidization the temperature shocks (7°C, 15°C, 38°C and 40°C) with different shocking durations (10-25 min for cold shocks or 15-60 sec for heat shocks) were applied at different times post-insemination (20-45 min) The results showed that the highest number (a cluster of 25-30%) of fertilized eggs synchronized development 20-30 min after incubation. At this time a high percentage (25-30%) of early metaphase and metaphase chromosomes could be observed. Shocking the eggs at 7°C or 40°C with longer durations resulted in decreased fertilization and Win normal fry (putative androgenetic P hypophthalmus). Variation in number of normal fry at hatching ranged from Oto 4% in fertilized eggs exposed to I 5°C or 38°C temperature shocks. Ten specimens of normal fry were examined by polymerase chain reaction using primers targeting a portion of the gene encoding the immunoglobulin heavy chain. Eight of these specimens exhibited the specific DNA band ( ~300 bp) of P hypophthalmus.
Nuclear DNA was isolated from the blood cells of catfish representing three families (clariidae, pangasiidae and ictaluridae) for analysis by polymerase chain reaction (PCR) and restriction enzymes. Primers specific for the CH4 exon of the gene encoding the immunoglobulin M heavy chain of channel catfish Uctalurus punctatus) were used. Nuclear DNA amplified with these primers yielded a single band of about 300 base pairs (bp) for Clarias macrocephalus, Pangasius gigas, Pangasius hypophthalmus and the hybrid of P. gigas X P. hypophthalmus. However, the same primers yielded two DNA bands of about 300 and 340 bp in Clarias gariepinus and in the hybrid of C. macrocephalus X C. gariepinus. Nucleotide sequences of the amplified DNA were determined for /. punctatus, C. macrocephalus, P. gigas and P. hypophthalmus. Based on the DNA sequence data, the restriction enzyme HpaI was used to further characterize the PCR products of P. gigas, P. hypophthalmus and their hybrid. Digestion with this restriction enzyme yielded one DNA band (300 bp) for P. gigas, two bands (100 and 200 bp) for P. hypophthalmus and three bands ( 100, 200 and 300 bp) for the hybrid. These findings would aid in identifying genetic contributions in hybrid, androgenetic, gynogenetic and polyploid catfish.
A primary cell line (designated as CCf) derived from caudal fin tissue of channel catfish, lctalurus punctatus, was developed using explant techniques. The cell line grew fastest in media supplied with FBS and channel catfish serum. The duplication time of the cell line under optimal conditions was ~56 h at a plating density of 1.1 x 105 cells/ml. The cell line has been propagated continuously for 25 passages (1:4 dilution per passage), cryopreserved, and recovered successfully at different passages. The cultured cells had fibroblastic morphology, and synthesized fibronectin and Type I and III collagens in the cytoplasm. The cell line maintained the normal diploid chromosome number (58) of channel catfish throughout the experiment. Nucleolus organizer regions were located on the short arms of a pair of medium-sized submetacentrics, which is typical for channel catfish. This study provides a method for acquiring a cell line from juvenile catfish without sacrifice, and is especially useful for early screening of valuable fishes.
The endangered razorback sucker Xyrauchen texanus is endemic to the Colorado River system in western North America and is threatened with extinction because of limited recruitment. To assist in management and recovery efforts, we developed methods for the cryopreservation of sperm, evaluated the influence of various factors on motility of thawed sperm, and examined the effect on fertilization of cooling rate and the addition of caffeine. Sperm samples cryopreserved with I 0% methanol (MeOH) had significantly higher postthaw motility than did samples preserved with 5% or 20% Me0H or with 5% or 10% dimethyl sulfoxide, N.N-dimethylacctamidc, glycerol, propylene glycol, or ethylene glycol. Sperm samples cryopreserved in 0.5- mL and 2.5-mL straws had significantly higher postthaw motility than did samples cryoprcserved in 0.25-mL straws. Exposure to 10% Me0H for up to 30 min did not significantly influence sperm motility before freezing or after thawing. Cooling rate (-21 °C/min or -91 °C/min) did not significantly influence sperm motility. Samples thawed in a water bath at 20°C, 30°C, or 40°C had significantly higher motility than did samples thawed on the laboratory bench ( 19°C). Refrigerated sperm had significantly higher motility after the addition of 0.005 M caffeine: however, caffeine did not increase the motility of thawed sperm. Fertilization percentage was 41 ± 31 % for the egg quality control treatments (fresh sperm) in the freezing rate study. The freezing rate of -91°C/ min yielded 66% fertilization relative to the control (actual value, 27 ± 26% ), which was significantly higher than the 12% fertilization (actual, 5 ± 3%) yielded by the freezing rate of -21°C/ min. Fertilization percentage was 25 ± 24% for the egg quality control treatments in the caffeine study. Caffeine-treated sperm yielded 60% fertilization relative to the control (actual, 15 ± 13% ), which was significantly higher than the I 6% fertilization (actual, 4 ± 4%) yielded by sperm without caffeine treatment.
Genetic research of fishes is hampered by lack of standardized karyotypes and reliable techniques of chromosome banding. The goal of this study was to develop a standardized karyotype of channel catfish lctalurus punctatus by a variety of handing techniques and computer-assisted analysis. Metaphase chromosomes were prepared from cultured leukocytes and kidney cells of adult and juvenile fish. Silver staining and alkali treatment methods were used to reveal nucleolus organizer regions (NOR) anti the location of constitutive hetcrochromatin (C-bands). Chromosomes were treated with IO different restriction enzymes, stained with Giemsa, and examined for banding patterns. The chromosomal data were analyzed with two computer software packages. The 29 chromosomes were classified into eight distinct groups based on morphology and size. The NOR were located on a pair of medium-sized submetacentric chromosomes (designated as D-11): this was consistent among cells from different specimens. The C-bands were small and restricted to centromeric regions and were useful for homologous pairing. A standard karyotype of CHG-handed chromosomes (C-bands visualized by treating with barium hydroxide and staining with Giemsa) was developed. The restriction enzyme Msc I produced informative banding patterns with dark telomeric bands and clear centromeric regions. The enzyme Hind III was most informative. yielding linear banding patterns that were consistent between members of homologous pairs. Our study provides a fundamental step in genome mapping of fishes. Standardization of the channel catfish karyotype and chromosome banding will facilitate physical mapping of genes in this important culture species.
To aid in artificial spawning of sciaenid fishes, the present authors developed techniques to collect, handle and cryopreserve sperm from red drum, Sciaenops ocellatus L. Sperm were collected by removing and slicing the testis, and adding Hanks' balanced salt solution (HBSS) or NaCl solution (each at 200-400 mOsm kg-1) as an extender. Sperm were activated with 800 mOsm kg-1 artificial sea water (ASW) to characterize motility. Sperm reached maximum motility (highest percentage motility observed for that sample) within 8 ± 1 s (mean ± SD) and remained at maximum motility for 33 ± 4 s. Sperm were exposed to graded osmotic pressures of ASW (8-800 mOsm kg-1) to determine the range of osmolalities that elicited motility. Threshold activation ( defined as ~ 10% motility) occurred at 351 ± 4 mOsm kg-1 and complete activation occurred at 5 3 9 ± 2 mOsm kg-1. Sperm stored at 200 mOsm kg-1 retained motility for up to 13 days. Dimethyl sulphoxide (DMSO) was used as a cryoprotectant at concentrations ranging from 7.5% to 15% (v:v) in HBSS (200 mOsm kg-1). There were no significant differences among postthaw motilities of sperm cryopreserved at any concentration of DMSO. Sperm thawed on the benchtop at 21 °C had lower post-thaw motility than did sperm thawed at 10, 20, 30, 40, 50 or 60°C in a water bath.
Optimization of conditions for short-term storage of gametes is important for the production of seedstock and genetic management of broodstock in aquaculture. We conducted a series of experiments to evaluate refrigerated storage of sperm of the Eastern oyster (Crassostrea viginica). Our objectives were to: 1) compare motility of oyster sperm suspended in artificial seawater (ASW) at five osmotic pressures (22, 203,403, 601, or 833 mOsm/kg) over 24 h; 2) compare motility of sperm suspended in solutions of ASW, Hanks' balanced salt solution (HBSS), or DCSB4 solution (all at 833 mOsm/kg) over days of refrigerated storage; 3) compare motility of sperm suspended in ASW or HBSS (833 mOsm/kg) in six ratios of sperm:extender (1:0, 1:1, 1:3, 1:7, 1:15, or 1:31) over 4 days of refrigerated storage, and 4) compare motility and fertilizing capacity of oyster suspended in ASW (200 and 830 mOsm/kg), ASW with 6% glycine (ASW+G), HBSS, and calcium-free HBSS (C-F HBSS) (all at 830 mOsm/kg). Significant differences (p < 0,001) were found in the motility of sperm suspended in ASW of different osmotic pressures. No significant differences (p = 0.267) were found in motility of sperm suspended ill ASW, HBSS, or DCSB4. Significant differences (p < 0.00]) were found in the motility of sperm suspended ill different ratios of sperm:extender. The highest motility was found in undiluted sperm and the lowest in the l :31 dilution for sperm suspended in ASW or HBSS. Significant differences (p = 0.0001) were found in motility of sperm suspended in various extenders. The highest motility was found in sperm diluted in CF-HBSS (96%), and the lowest in sperm diluted in ASW al 200 mOsm/kg (12%). The highest percent fertilization (48%) (as measured by larval development at 12 h) was obtained when eggs were fertilized with sperm diluted in C-F HBSS. These results indicate that for storage (4 days), it is best to leave sperm samples undiluted. However, when sperm samples are diluted for use within 24 h, it is best to maintain high sperm concentrations and to use C-F HBSS as an extender.
Channcl catfish lctalurus punctatus arc commonly spawned for research purposes by pairing of a hormonally treated female with a male in flowthrough aquaria. A technique that allows hormonal induction of ovulation in females without pairing would accelerate genetic improvement and production of hybrid catfish. Over a 3-yr period (1994, 1995, and 1996) we conducted a series of trials to demonstrate the potential for artificial spawning in recirculating systems, and in 1996 we included trials with grouped females in addition to male-female pairs. Females were induced to spawn with injection of synthetic leuteinizing hormone--releasing hormone, and those that ovulated were stripped and the eggs were artificially fertilized. During 1994 and 1995, all fish were spawned hy pairing, and in 1996, half of the females were spawned by pairing and half were grouped in tanks without males. Spawning success (percent of females that produced eggs), latency (time between injection and ovulation), and percent fertilization were observed for the paired and grouped trials. Spawning success was 36% in 1994 (N = 36), 22% in 1995 (N = 54), 41% in 19% (N °= 27), and 58% for grouped females (N = 26). The latency period was 113 +/- 69 h in 1994, 109 +/- 57 h in 1995, 44 +/- 8 h in 1996, and 50 +/- 9 h for grouped females, Percent fertilization was 16 +/- 26% for eggs stripped in 1994, 72 +/- 25% in 1995, 43 +/- 20% in 1996, and 16 +/- 37% for grouped females. In 1995, water quality problems were associated with high mortality of females (24 of 44 females; 4 of 44 males). The metabolic demands of final oocyte maturation in combination with methemoglobinemia caused by high nitrite levels could account for the increased vulnerability of females. These trials indicate that with adequate biofiltration, artificial spawning is possible in recirculating systems and with females grouped rather than paired. Further research on hormone dosage and timing of egg stripping will increase the utility of grouped spawning of channel catfish.
A replication banding procedure using fluorouracil (FU) and bromodeoxyuridine (BrdU) was developed for use with cultured leukocytes of channel catfish (lctalurus punctatus). Analyzable banding patterns were produced on chromosomes stained with a fluorochrome plus Giemsa method. The consistency of the chromosomal bands was evaluated by computer-assisted image analysis. Banding patterns of representative chromosomes including those bearing nucleolus organizer regions (NOR) were reproducible. A standard RBG-banded karyotype (R bands by BrdU and Giemsa) was established with idiograms of each chromosome. We also developed procedures to study sister-chromatid exchange and sister-chromatid differentiation in cultured leukocytes, without addition of mutagenic substances. The average occurrence of SCEs in the absence of mutagens was 3.0 +/- 1.0 (n 60) chromosomes per cell and was not significantly different among fish (P .26).
The activity of an insect promoter of the cecropin B gene (Cec B) was investigated using green fluorescent protein (gfp) as a reporter in cells of channel catfish (Ictalurus punctatus). The expression vector pQZ-1 containing the Cec B promoter and a modified gfp cDNA sequence was delivered by lipofection to three catfish cell types: fibroblast and leukocyte cell lines, and primary cultures of leukocytes. No resistance genes were included in the vector for selection of GFP-expressing cells. The GFP mRNA was detected in all three cell types with 5 to 10 times higher concentrations observed in leukocytes than in fibroblasts. Expression was enhanced with the addition of irradiated Flavobacterium columnare (7.0 x 106 cells/ml) or Escherichia coli LPS (125 µg/ml ). Quantitative RT-PCR showed GFP mRNA reached maximum levels 24 h after bacterial challenge in fibroblast cells, and at 10-12 h after LPS challenge in fibroblasts and leukocytes. The number of fibroblasts expressing GFP increased by 0.8%, and the average of green fluorescence intensity increased by 52.8%, whereas the increase in leukocytes was 0.13% in cell number and 3.4% in fluorescence intensity. These results suggest that the transcription of the Cec B promoter in channel catfish cells exhibited an inducible pattern and could be placed under the control of the immune system (in vivo). The mechanisms for endogenous activation of the Cec B promoter and for production of gfp RNA in unchallenged cells remain to be studied.
The homologous sequences of human or mouse SOX1, SOX4 and SOX11, and one novel Sox gene (named Ccf-SoxN) were identified in the genome of channel catfish Ictalurus punctatus. Identification of these genes is a potential step in understanding development regulations including sex determination in channel catfish.
A number of fish species have declined in population size or become extinct in the United States, primarily
as a result of habitat changes (Ono et al. 1983; Minckley and Deacon 1991; Cloud and Thorgaard 1993;
Magnuson et aL 1996). One approach that has been considered in some cases where drastic declines have
occurred is the use of gene banking based on sperm cryopreservation to avoid the loss of valuable genotypes.
However, such efforts have been relatively limited to date. This paper reviews some of these efforts and
identifies some factors that have limited the application of this conservation approach in the United States.
Government in the United States is relatively decentralized compared to that in many other
countries. The national (federal) government has responsibility for anadromous and marine fishes and
for enforcement of some national regulations (e.g., the US Endangered Species Act). Relevant federal
agencies include the US Fish and Wildlife Service, which is primarily concerned with freshwater fishes,
and the National Marine Fisheries Service, concerned with marine and anadromous fishes. The 50 States
also have authority over fishes within their borders. Examples of State management agencies in the
northwestern United States include the Washington Department of Fish and Wildlife and the Idaho Department
of Fish and Game. Indian tribes within the United States have their own governmental authority and
frequently have their own fishery management agencies and biologists. A given fish population may thus
be subject to management by several agencies. Management of the salmon of the Columbia River is a prime
example of this overlapping responsibility. This rather complex situation does not always contribute
to the most efficient management of the populations.
Given the size of the United States and the complexity of the web of agencies managing fishes
in this country, we likely may not have identified the full scope of gene banking efforts for fishes
in the United States in this review. However, in discussing this issue with people actively engaged in
such efforts we believe that we have identified the major focus areas and programs for endangered fishes.
The three major foci of gene banking efforts in the United States appear to be (1) Columbia River salmon,
(2) Colorado River fishes, and (3) Midwestern and Eastern US sturgeon. We will review each of these foci
in order and then discuss some of the overall issues and constraints common to these efforts.
Techniques were developed for holding and conditioning of eastern oysters, Crassostrea virginica (Gmelin), in a recirculating system. Oysters collected in February from public oyster grounds off the coast of Louisiana were maintained in a recirculating system for 8 wks. For conditioning, water temperature in the system was gradually raised with a heat pump from l4°C, and held at 25°C for 6 wks. Oysters were fed a diet of algal paste (lsochrysis galbana for the first 6 wks and Chaetocerus calcitrans for the last 2 wks). Water quality, mortality, Perkinsus marinus infection, gonad development, and physiologic condition (dry tissue-to-dry shell ratio, dry tissue-to-wet tissue ratio, digestive diverticula tubule ratio) were monitored. At weeks 7 and 8, the laboratory-held oysters were compared with field controls held at Grand Isle, Louisiana. Water quality in the system remained within target ranges. Mortality was low (18 of 300 oysters stocked) and not associated with P. marinus infection. In the laboratory at week l, the gonads of all oysters sampled were classified as immature or in early development. By week 5, the gonads of 73% of oysters sample.d were classified as mature. Physiologic condition decreased in the laboratory. Field controls reached a higher mean gametic stage and were in better physiologic condition at the end of the 8-wk study. These differences were attributed to differences in nutrition available between the field and laboratory. This study demonstrated that conditioning of Crassostrea virginica is possible in a closed. recirculating system, although improvements in nutrition would be useful.
An in situ polymerase chain reaction (ISPCR) procedure was developed for analysis of single-locus genes of the channel catfish (lctalurus punctatus). Intact leukocytes, suspended in phosphate buffered saline (1.0 x 10^6 cells/milliliter), were distributed on microscope slides and digested with proteinase K (6 µg/ml) for 1 h at 27°C. The PCR was performed on the slides with a thermal cycler (PTC-100, MJ Research, lnc., Watertown, MA). Primers were designed to target the gene Ig H. encoding the constant region of the immunoglobulin heavy chain. After amplification with biotin-labeled primers, the intracellular PCR products were detected by color development or fluorescent methods. The cells on treatment slides were strongly positive for the target gene. This was verified by use of an indirect procedure, in which the ISPCR was performed using unlabeled primers, followed by use of a biotin-labeled probe. The ISPCR technique was also applied to the location of the gene in interphase nuclei. Two copies of the target gene were revealed in positive interphase nuclei. For validation of the TSPCR technique, we performed the technique on a mixture of channel catfish leukocytes and fetal donkey dermal cells. More than 90% of the catfish cells exhibited fluorescence associated with the presence of the target gene, whereas no fluorescence was observed on the fetal donkey dermal cells. Liquid-phase PCR was also included as a positive control in each experiment and yielded products of the expected size (303 base pairs) and DNA sequence. Localization of a single-locus gene by ISPCR is new to nonmammalian species and will provide an important tool for gene mapping and transgenic studies in fishes.
Establishment of chromosome identity is the first step of physical genome mapping. This step is hindered by a lack of banding techniques and size homogeneity in the chromosomes of the eastern oyster, Crassostrea virginica. In this study, chromosomes prepared from adult gill, ripe gonad tissues, and embryos were analyzed with a computer-based measurement system. Chromosomes from embryos were elongated with excellent morphology and identifiable secondary constrictions, although homologous pairs were difficult to establish because of asymmetric sizes found between homologues. Meiotic chromosomes at the stages of diakinesis (testis) and pachytene (ovary) offered distinct advantages for karyotyping. These chromosome bivalents possessed a haploid chromosome number (n = JO) with transverse chromomere bands analyzable by microdensitometry. Chromosomes derived from gill tissue were highly condensed and few spreads were analyzable. Idiograms of each chromosome were developed in this study based on size, centromere position, and chromomere bands. These results indicate that mitotic and meiotic cells are each important for the study of chromosomes of the eastern oyster and that computer-assisted analysis will be useful for establishment of karyotypes and idiograms.
At present. establishment of a cell line from bivalve molluscs has been unsuccessful, and in vitro work is limited to primary cell cultures. We sought to improve attachment and spreading of cells ol' the eastern oyster, Crassostrea virginica, lo aid primary cultures and to assist development of a bivalve cell line. Our objectives were to examine the effects of substrate on ventricle cell viability, attachment, and spreading by testing of collagen I, collagen IV, fibronectin, laminin. poly-D-lysine, and two types of uncoated tisBuc culture plates (Falcon® and Corning®). Experiments were conducted by incubating cells with the various substrates for 24 h and 5 d. An assay with a telrazolium compound (MTS) was used to estimate cell numbers based on metabolic activity. Although differences in MTS assay values for substrate effect on cell viability were detected at 24, h and at 5 d (P > 0.0001), these were attributed to variations in metabolic activity due to different levels of attachment and spreading among treatments. Differences among treatments were detected in attachment and spreading at 24 h and 5 d (for all, P > 0.0001). At 24 h, poly-D-lysine induced the highest levels of attachment and spreading; no other factor performed better than the uncoated Falcon® substrate, and collagen l performed most poorly. At S rl, poly-n-lysine and the uncoated Corning® substrate induced significantly higher level:-; of attachment and spreading than did the uncoated Falcon@ substrate, and collagen l performed most poorly. From these result:;, poly-D-lysine best promoted cell attachment and spreading. Fibronectin (at 24 h) and laminin (at 5 cl) warrant further study. Along with improvements in medium composition, future work should involve screening of other attachment factors and combinations of factors, including those of bivalve origin.
The physical location of the 28S ribosomal RNA gene (28S rDNA) was localized to the short arm of chromosome number 1 of the eastern oyster ( Crassostrea virginica) by fluorescence in situ hybridization. The existence of a single locus of the 28S rDNA in the genome of the eastern oyster was concluded based on the findings from metaphase chromosomes prepared from gill, mantle, and embryos and from meiotic chromosome bivalents prepared from ripe gonad tissue. The region of the chromosome occupied by the gene was found to be GC-rich, and the location of probe DNA used to identify the 28S gene corresponded exactly with the location of the nucleolus organizer region. The transcriptional activity of the gene did not vary among different cell types but changed with different phases of mitosis. This study is the first report of physical mapping of a specific gene in mollusks and provides techniques for detection of integration of foreign DNA in the oyster genome.
Genome analysis is an essential step for genetic mapping of economically important ictalurid catfish species and provides basic knowledge for breeding and phylogenetic studies. lil this review article, we summarize results of studies on nuclear DNA cont,cnt, chromosome number and structure, karyotype standardization, and physical genetic mapping of catfishes and their hybrids. The genome sizes of four species of ictalurid catfishes representing the genera lctalurus, Ameiurus, and Pylodictis were 2.11 ± 0.05 picograms (pg) ranging from 2.06 pg (lctalurus furcatus) to 2.20 pg (Ameiurus melas) and with a variation of less than 2.5% within a species. This low variation may relate to a low occurrence of constitutive heterochromatin in catfish chromosomes. Nuclear DNA and chromosomes were found to segregate precisely as a function of haploid levels and were stable in genomes of interspecific or intergeneric hybrids. ldentification of individual chromosomes has been achieved by different techniques including differential staining for nucleolus organizer regions and heterochromatin, and by restriction enzyme banding and replication banding. Standard karyograms were produced with the assistance of a computer-based image analysis system for morphometric measurements and location of chromosome hands. In situ polymerase chain reaction has been used to identify the chromosomal location of a gene existing in single copy in the haploid genome. which provides a promising method for the physical mapping of catfish genomes. However, supporting techniques must he developed lor analysis of chromosome identity after hybridization treatments.
In recent years aquaculture has become an important sector of fish production in the region of the Mekong River. Catfishes of the families Pangasiidae and Clariidae are commercially produced throughout the region. Indigenous species include the Chao Phraya catfish, or swai, Pangasius hypophthalmus (formerly P. sutchi) (Roberts and Vidthayanon 1991), the Mekong giant catfish, or pla buk, Pangasius gigas (formerly Pangasianodon gigas), the walking catfish Clarias batrachus and Clarias macrocephalus. The Mekong giant catfish is the world’s largest freshwater catfish, often surpassing 250 Kg in weight (Figure 1). This fish is of considerable popularity in Thailand, Laos and Cambodia and has been the object of a traditional capture fishery for centuries. Abruptly declining harvests have indicated that the Mekong giant catfish is an endangered species. The Thai Fisheries Department initiated an artificial breeding program for wild-caught Mekong giant catfish in 1983 and produced 200,000 fry which were reported to have attained an average of 21 Kg in 4 yr in earthen ponds (Pholprasith 1996). Appropriate aquaculture techniques have been developed for P. gigas and hybrids (P. hypophthalmus x P. gigas). Moreover, aquaculture production of commercial scale is in the expansion stage.
In aquaculture, cryopreservation studies have primarily addressed spermatozoa of three groups of commercially
important fishes (salmonids, tilapias and carps) (Stoss 1983). However, work with larvae of fish or
other organisms such as mollusks is scarce (Gwo 1995). In mollusks, the Japanese oyster Crassotrea
gigas has been the species most used to study cryopreservation of larvae (Table 1). Other species of
oysters, including those of commercial importance, have not been studied. Cryopreservation of oyster
gametes and larvae has been tested only in the laboratory, and has yielded only a single report of
oyster growth beyond planktonic stages (Paniagua-Chavez et al. 1998a). Given the benefit that this
technique offers to research and the commercial oyster industry, cryopreservation of oyster gametes and
larvae should be developed for application in the hatchery.
In the United States the most important oyster is the eastern oyster Crassostrea virginica. In
Louisiana the production reached a value of $49,000,000 in 1998 (Avery et al. 1999). Along the Atlantic
and Gulf coasts, production of eastern oysters has declined due to a variety of reasons including a lack
of consistent seed supply, excessive harvest, disease and natural predation (Supan and Wilson 1993).
The production of cryopreserved gametes or larvae would improve hatchery production of seedstock oysters,
thus allowing distribution of improved or genetically modified stocks.
This study represents the first successful production of seedstock from cryopreserved larvae
of any aquatic food organism cultured for human consumption
(Paniagua-Chavez et al. 1998a) and it documents for the first time the production of oyster seedstock
from eggs fertilized with thawed sperm.
As cryopreservation of fish sperm becomes increasingly applied, it creates new opportunities for genetic
study of fishes. However, methods for the isolation of DNA from cryopreserved fish sperm have received
only limited study (e.g. Cummings and Thorgaard 1994). Procedures for cryopreservation of fish sperm
involve the use of cryoprotectant molecules and specific cooling rates to control the formation of ice
crystals and the level of dehydration in cells during freezing. It is important to evaluate the effect
of cryopreservation procedures on the purity and yield of DNA from fish sperm with respect to the long-term
value of samples stored in germplasm repositories.
Cryopreserved sperm can be used for artificial spawning, and genetic analysis of frozen samples
would be useful in marker-assisted broodstock selection for genetic improvement of aquaculture species.
Cryopreserved sperm allows conservation of genetic resources in endangered species and control of genetic
diversity in artificial propagation programs. Identification of rare alleles in frozen sperm could
provide genetic markers for monitoring of stock enhancement programs in wild fishes. Isolation of DNA
from cryopreserved sperm offers verification by genetic analysis of the source of sperm from high-value
fish such as koi carp Cyprinus carpio that can be worth thousands of dollars apiece based on coloration
and markings.
Therefore, this study was designed to develop procedures for DNA isolation from fish sperm
cryopreserved for use in fertilization (Pittman-Cooley and Tiersch, 1999). Our objectives were to: 1)
evaluate the effect of cryoprotectants, freezing rate, and storage temperature on the purity and yield
of DNA isolated from cryopreserved sperm of channel catfish Ictalurus punctatus; 2) evaluate utility of
the isolated DNA for use with the polymerase chain reaction (PCR), and 3) evaluate DNA isolation procedures
for use with cryopreserved sperm of other fishes.
Few studies have addressed sperm cryopreservation in aquarium fishes (body sizes of 10 cm or less). There are several challenges inherent in developing cryopreservation procedures for these fishes. First, their small body size and sperm volume limit experimental replication and the numbers of treatments possible without. pooling of samples. This hinders research especially if many experimental variables arc evaluated. The small sample volume necessitates identification of optimal sperm-to-egg ratios to maximize fertilization potential, and places greater emphasis on increasing and maintaining sperm viability after thawing. Other technical problems include the use of 0.25-ml French straws which increase difficulties in sample handling (automated straw fillers are more common for the 0.5-ml straw) and labeling. Sperm cryopreservation or live-bearing fishes (with internal fertilization) is essentially unexplored. The sperm of these fishes are sufficiently different in structure ( e.g., head shape) and physiology ( e.g., energy metabolism) from the sperm of other fishes that the need to develop specialized techniques is almost assured. The requirement for artificial insemination also introduces a new variable complicating the collection of data (e.g., assessing fertilization is not straightforward with internally held eggs). Cryopreservation in aquarium fishes will assist the development ( e.g., through selective breeding), protection ( e.g., through germplasm repositories), and distribution (e.g., through shipment of frozen sperm) of research lines, and offers benefits for restoration of endangered species.
The native fishes of the Colorado River system of the southwestern United States have declined
substantially during the past century and their prospects for survival are not good. Numerous
factors including introduction of exotic species and construction of dams have extensively disrupted the
natural habitat of these fishes and has resulted in major changes in the aquatic fauna. The razorback
sucker Xyrauchen texanus was once abundant and widely distributed throughout the Colorado River and its
tributaries, but is now found commonly only in the middle Green River and Lake Mohave, below Hoover Dam
(Minckley et al. 1991, Modde et al. 1996). Razorback suckers are threatened with extinction because of
limited recruitment into the adult size classes, and in 1991, received a formal listing under the Endangered
Species Act (U.S. Fish and Wildlife Service 1991). Because of the lack of natural recruitment,
artificial propagation and reintroduction are emphasized for the conservation, management and recovery of
this species. Methods that enable the transfer of gametes from wild populations to hatchery broodstock
(e.g. Cloud et al. 1990) are needed to aid in this recovery program.
Recommendations identified by a multi-agency management team include the stocking of Lake Mohave
with razorback suckers to replace the aging population (Figure 1). Razorback suckers are currently
maintained for research and stocking at the U.S. Fish and Wildlife Service (USFWS) Dexter National Fish
Hatchery and Technology Center (Johnson and Jensen 1991) and at the Willow Beach National Fish Hatchery
(WBNFH). Management of razorback suckers has included production and distribution of fertilized eggs and
larval fish in protected backwaters adjacent to Lake Mohave (Mueller 1995). Storage and cryopreservation
of sperm is an effective management tool for conserving genetic resources of threatened and endangered
populations. This technology offers advantages by providing genes from wild populations for hatchery
broodstock, greater control in breeding programs, and the ability to store large amounts of valuable
germplasm for long periods. Gamete storage is an effective way of solving hatchery-related problems of
differential maturation of broodstock by allowing flexibility in spawning time. Our purpose in this work
was to improve and integrate gamete collection, storage and cryopreservation for enhancement of recovery
efforts for razorback suckers (Carmichael et al. 1996, Figiel et al. 1996, Tiersch et al. 1997, 1998).
The objectives were to: 1) develop methods for collection of sperm allowing integration with established
sampling programs; 2) characterize sperm motility and duration; 3) develop methods for refrigerated storage
of sperm; 4) develop methods for the cryopreservation of sperm; 5) fertilize eggs with cryopreserved sperm,
and 6) investigate methods for incubation of eggs.
In this section, we have chosen to provide a single integrated set of methods as a starting point for development of protocols and standardization of reports. These methods have been adapted for sperm cryopreservation of more than 30 species of marine and freshwater fishes in our laboratory. The information provided here can be supplemented with methods and information in other chapters to develop new protocols or to work with previously unstudied fishes. There is also a large amount of practical information available for topics such as cryopreservation and freeze-drying methods (Simione and Brown 1991, Day and McClellan 1995), cryopreservation of algae (Morris 1981) and semen of boars (Johnson and Larsson 1985, Johnson and Rath 1991). Sources such as these can be valuable for developing and using cryopreservation protocols even though they are not intended to address aquatic species. Overall, there are several basic components involved in development of cryopreservation protocols. These will be covered individually in the following sections: 1) Sperm collection; 2) Motility estimation; 3) Extenders and refrigerated storage; 4) Labeling of straws, goblets and canes; 5) Addition of cryoprotectants; 6) Filling of straws; 7) Freezing procedures; 8) Storage procedures, and 9) Thawing and fertilization. Each component is important, and just as links form a chain, failure of any single component can lead to failure for the entire project.
Unlike studies in biomedical fields, research in aquatic species is often hindered by the lack of
pre-existing reagents, tools and methods. Research tools such as cell lines, DNA probes and tagged
antibodies that are readily available, indeed taken for granted, for work in mice or humans, do not
exist for aquatic species. This is especially true for genetic analysis of even commercially important
aquaculture species. For example, it is well established that the quality of chromosome preparations
is directly related to the quality of source material. For this reason, reproducible cell lines are
often used as a source for high quality chromosomes. In mollusks such as oysters, there is no single
cell type that possesses all traits required for use in chromosome studies (Zhang et al. 1999).
Somatic tissues such as mantle and gill of adult oysters are available year-round and provide chromosomes
with minimal background. However, most adult tissues of oysters have low mitotic activity, and
treatments for stimulation of mitotic activity in oysters are not available (Cornet 1993).
For such reasons, it has been extremely difficult to establish cell lines to support studies
such as karotyping (Buchanan et al. 1999), and only a single cell line has ever been developed for mollusks
(Hansen 1976). Meiotic chromosomes prepared from gonad tissue of oysters during spawning season are
helpful for identification of individual chromosomes due to the presence of unique structures
(chromomeres), however these chromosomes are not useful for karyotyping because of their diffuse
and overlapping appearance (Figure 1). The embryos of oysters can provide actively dividing cells,
and chromosomes prepared from these cells have morphology useful for mapping of gene location.
Chromosomes at different phases of division can be found in these cells, and the elongated prophase and
pro-metaphase chromosomes are most suited for application in high-resolution genome mapping (Table 1).
However, embryonic cells are only available during spawning season. Recent studies have shown
that larvae of the eastern oyster Crassostrea virginica can be cryopreserved and remain viable after
thawing (Paniagua-Chavez et al. 1998 and pp. 230-239, this volume), circumventing the problem of
seasonal availability of embryos. In channel catfish, we have used cryopreservation to store primary
cultures of leukocytes for chromosome analysis after thawing (Zhang and Tiersch 1995). Our goal in
this study was to develop procedures for obtaining chromosomes from cryopreserved larvae of the eastern
oyster and to establish a year-round source of material for preparation of chromosomes for physical
genome mapping despite the lack of cell lines.
Fish sperm cryopreservation is documented by a large body of technical research. However, there are no reports of the economic requirements for using this technology in aquatic species. This study establishes a generic analysis of the investment and operating costs required to integrate sperm cryopreservation into existing fish hatcheries and can serve as a template for implementation of cryopreservation programs. Equipment and supplies were identified in a species-independent description of sperm cryopreservation, and capital and operating costs were documented for private and public hatcheries at three production capacities (3,000, 6,000, and 9,000 0.5-mL straws). Compared to public hatcheries, investment costs were found to be 70% higher, and operating costs 20% higher for private hatcheries due primarily to interest on borrowed capital. Equipment costs were dependent on the scope of work. Investment in required equipment incurred costs of $5,460 to $10,458 (public) or $9,497 to $18,190 (private), depending on production level. Purchase of optional research equipment increased initial investment by 300% without increasing output. Per unit costs decreased at higher production levels for all scenarios, but greater economies of scale were associated with private research hatcheries. Production costs ranged between $6.13 and $1.86 per straw (private), to $1.59 to $1.18 per straw (public). Increased commercialization is expected to occur as research protocols for fish sperm cryopreservation are applied in the private sector and markets for cryopreserved sperm are established.
This study describes the use of the polymerase chain reaction for physical mapping of fish genes. A 287-base pair (bp) fragment of the 28S ribosomal RNA gene (28S rDNA) of channel catfish lctalurus punctatus was isolated and sequenced with human-derived primers. The nucleotide (nt) sequence of this fragment was 20 bp shorter than that of the corresponding region of the human 28S rDNA. The gene was mapped to chromosomes of channel catfish by fluorescence in situ hybridization (FISH) and in situ polymerase chain reaction (ISPCR). A major locus and a minor locus of 28S rDNA were found on chromosomes of channel catfish. The major locus was associated with the active nucleolus organizer region (NOR) sites. The minor locus was highly resolved and not detectable by silver staining, suggesting that this locus was not involved in synthesis of ribosomal RNA and possessed fewer copies of 28S rDNA. Both loci contained GC-rich DNA clements that could be components of 28S rDNA repeated units. In this study, a potential method of comparative mapping of the channel catfish genome has been presented by using human-derived oligonucleotide sequences. These data demonstrate that ISPCR is highly specific and will be useful in physical mapping of fish genomes.
Ionizing radiation has been used for decades in studies involving aquatic organisms to produce androgenesis and gynogenesis and in attempts to produce reproductively sterile individuals. Tn these studies, gametes or organisms were exposed to a broad range of doses of ionizing radiation in various containers to obtain the desired results. However, there are many factors that affect irradiation levels and cause target doses and actual absorbed doses to be different. Our goal in this study was to use intensive dosimetry to determine the variation in dosage possible in irradiation of aquatic organisms. Using the Fricke dosimetry technique, we found suprisingly high levels of vertical and horizontal variation in dose rates inside of a small (hut typical) container ( 14.5 cm high, 16.7 cm wide, and 2.8 Lin volume). The presence or absence of container rotation further affected these variations. When the container was rotated, vertical variation ranged between 1% and 21 %, and horizontal variation ranged between 10% and 19%. When the container was not rotated, vertical variation ranged between 6% and 28%, and horizontal variation ranged between 20% and 72%. Comparisons between air and water as a surrounding medium showed that the dose rate in air could he 42% higher than in water when the container rotated and as much as 218% higher in air when the container did not rotate. These variations would cause samples irradiated in water to receive dosages lower than desired. It is therefore crucial that careful dosimetry be performed before carrying out experiments and that they be performed in the same medium in which specimens are to be irradiated (presumably water for aquatic organisms). Such measurements are typically not reported or are not performed in irradiation studies of aquatic organisms. Experimental errors such as these would complicate comparisons among studies and organisms and hinder development and application of valuable techniques such as radiation-induced sterilization of genetically modified organisms.
Improved techniques for storage and evaluation of fish sperm would enhance breeding programs around the world. The goal of this study was to test the effect of antibiotics on refrigerated sperm from Nile tilapia (Qreochromis niloticus) by use of flow cytometry with 2 dual-staining protocols for objective assessment of sperm quality. Concentrations of 1 x Hf_ sperm/mL were suspended in Ringer's buffer at 318 mOsmol/kg (pH 8.0). The fluorescent stains Sybr 14 (10 µM), propidium iodide (2.4 mM), and rhodamine 123 (0.13 µM) were used to assess cell viability and mitochondrial function. Three concentrations of ampicillin, gentamicin, and an antibiotic/antimycotic solution were added to fresh spermatozoa. Motility estimates and flow cytometry measurements were made daily during 7 d of refrigerated storage (4 °C). The highest concentrations of gentamicin and antibiotic/antimycotic and all 3 concentrations of ampicillin significantly reduced sperm viability. The highest of each of the 3 antibiotic concentrations significantly reduced mitochondrial function. This study demonstrates that objective sperm quality assessments can be made using flow cytometry and that addition of antibiotics at appropriate concentrations can lengthen refrigerated storage time for tilapia spermatozoa. With minor modifications, these protocols can be adapted for use with sperm from other species and with other tissue types.
Conditions were developed to cryopreserve cells from pronase-dissociated atria and ventricles of eastern oysters (Crassostrea virginica). The effect of three concentrations (5. 1O. 15%) of the cryoproteclants (dimethyl sulfoxide. glycerol. and propylene glycol). three thawing temperatures (25. 45. 75° C). and three cooling rates (slow. medium. fast) were compared. Cells were frozen at -80° C and plunged in liquid nitrogen. Thawed cells were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound. 3-(4.5- dirnethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium. and by comparing the relative spreading of cells between treatments. The best conditions for freezing and thawing of cells for each cryoprotectant were selected and a final study was performed to compare cryoproteetants. For this final study. we measured the number of cells and their viability 3 d after thawing. in addition to determining cell metabolic activity and cell spreading. Primary cultures of cells frozen without cryoprotectant and of nonfrozen cells were used as controls in all studies. Atrial cells were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate. and thawing at 45° C. After thawing. atrial cells showed 53 ± 5% of the metabolic activity. 84 :': 5c70 of the number. and 92 ± 2% of the viability of nonfrozen cells. For ventricular cells, 10% glycerol, a medium cooling rate, and thawing at 25° C yielded the best results. The thawed ventricular cells showed 83 +/- 5% of the metabolic activity. 91 ± 5% of the number. and 96 ± 2% of the viability of nonfrozen cells.
Previous reports on viability of cryopreserved oyster larvae have been limited to evaluation of planktonic stages for less than 6 d after thawing. However, for hatchery and aquacultural purposes the performance of larvae through settlement is of primary importance. The present experiments on production of oysters from cryopreserved larvae were conducted in two consecutive years. Observations through veliger larvae only were collected from the first year because of interruption by a hurricane. In the second year, growth was observed for 4 months. A total of 3 x 106 trochophore larvae (12 h post-fertilization) were diluted in a cryoprotectant solution composed of artificial sea water and 15% propylene glycol. Larvae were placed in 5-mL straws, frozen at -2.5 °C per min, and stored in liquid nitrogen. The frozen material was transported to an oyster hatchery at Grand Isle, Louisiana. Larvae were thawed at 70 °C for 15 sec and were incubated in a 200-L tank. For a control treatment, 6 x 106 eggs were fertilized and incubated in the same conditions as the thawed larvae. After 24 h of incubation, 28% of the control group developed into D-stage larvae, while 24% of the thawed larvae developed to this stage. Larvae were fed daily with 20 Lor microalgae (Isocrysis galbana) for 10 d. After incubation, larvae were allowed to metamorphose and attach to substrate and were held for 8 d. Spat were placed in mesh plastic bags and suspended in Caminada Bay (29° 15' 12"N, 90°03'26"W) to evaluate survival and growth. After 4 months, 1,000 oysters from the control group, 850 from the thawed larvae, and 57 from naturally spawned oysters were found. The results indicate that oysters produced from thawed larvae developed normally in the hatchery, opening opportunities for use of cryopreserved larvae in oyster research and in commercial industry.
To evaluate the potential for fertilization by sperm injection into fish eggs, sperm from zebrafish, Danio rerio, were microinjected directly into egg cytoplasm of two different zebrafish lines. To evaluate physiological changes of gametes on the possible performance of intracytoplasmic sperm injection (ICSI), four different combinations of injection conditions were conducted using activated or nonactivated gametes. From a total of 188 zebrafish eggs injected with sperm in all treatments, 31 (16%) developed to blastula, 28 (15%) developed to gastrula, 10 (5%) developed abnormally to larval stages, and another 3 (2%) developed normally and hatched. The highest fertilization rate (blastodisc formation) was achieved by injection of activated spermatozoa into nonactivated eggs (35%). Injections were most effective when performed within the first hour after egg collection. Flow cytometric analysis of the DNA content of the developing ICSI embryos revealed diploidy, and the use of a dominant pigment marker confirmed paternal inheritance. Our study indicates that injection of a single sperm cell into the cytoplasm of zebrafish eggs allows fertilization and subsequent development of normal larvae to hatching and beyond.
There is a need for research in disease resistance and microbial elimination in the eastern oyster Crassosostrea virginica. Gene transfer may lead to advances in this area, and a means of selecting transfected larvae would be useful. We transfected 3-hour-postfertilization embryos with the bacterial gene aminoglycoside phosphotransferase II (neo'), which confers resistance to neomycin and related antibiotics such as G418. The antibiotic G4 l 8 was examined as a potential selective agent. A neutral red assay was used to determine survival after 48 hours of exposure to various concentrations of G418 (0-4 mg/ml). We examined the effects of electroporation and chemically mediated transfection of 3-hour-postfertilization embryos on survival to straight-hinge larvae. DNA alone was found to have no effect on survival (P > .05). For electroporation we found that increased voltage and pulse duration decreased survival (P < .05). Chemically mediated transfection did not significantly affect survival (P .5172). Transgenic larvae were identified after electroporation and chemically mediated transfection. These larvae were reared for 24 hours and exposed to G418 at 0.3 mg/ml for 48 hours. Significant differences in survival between transfected and nontransfected larvae were detected for electroporation (P .0147) and chemically mediated transfection (P = .037). Gene transfer was also confirmed with polymerase chain reaction and observation of expression of green fluorescent protein. This study documents the first successful insertion and expression of foreign DNA in eastern oyster larvae.
Few studies have addressed sperm cryopreservation in aquarium fishes (body sizes of 10 cm or less). There are several challenges inherent in developing cryopreservation procedures for these fishes. First, their small body size and sperm volume limit experimental replication and the numbers of treatments possible without pooling of samples. This hinders research, especially if many experimental variables are evaluated. The small sample volume necessitates identification of optimal sperm-to-egg ratios to maximize fertilization potential and places greater emphasis on increasing and maintaining sperm viability after thawing. Other technical problems include the use of 0.25-ml French straws, which increase difficulties in sample handling (automated straw fillers are more common for the 0.5-ml straw) and labeling. Sperm cryopreservation of live-bearing fishes ( with internal fertilization) is essentially unexplored. The sperm of these fishes is sufficiently different in structure (e.g., head shape) and physiology (e.g., energy metabolism) from the sperm of other fishes that the need to develop specialized techniques is almost assured. The requirement for artificial insemination also introduces a new variable complicating the collection of data (e.g., assessing fertilization is not straightforward with internally held eggs). Cryopreservation in aquarium fishes will assist the development (e.g., through selective breeding), protection (e.g., through germplasm repositories), and distribution (e.g., through shipment of frozen sperm) of research lines and offers benefits for restoration of endangered species.
The eastern oyster Crassostrea virginica provides a commercially valuable industry along the eastern and Gulf coasts of the United States. Recently this industry has been damaged by disease problems, creating an interest in the use of gene transfer (transfection) to improve disease resistance. We transfected adult oysters with two genes, red-shifted green fluorescent protein (rsGFP), commonly used as a reporter gene, and the lytic peptide cecropin B (cepB), known to have antimicrobial properties. Oysters were transfected by injecting DNA mixed with SuperFect® reagent (Qiagen Inc.) into the adductor muscle sinus. Oysters were assigned to three groups of 15: the first was injected with rsGFP complexed with transfecting reagent; the second was injected with cepB complexed with transfecting reagent; and the third was injected with saline (control group). Hemolymph was collected at 4 and 10 d after injection. DNA was extracted for analysis by polymerase chain reaction (PCR), and hemocytes were examined by flow cytometry and fluorescence microscopy for detection of green fluorescence due to rsGFP expression. The rsGFP gene was detected by PCR in hemocytes from 14 of 15 oysters at day 4, and in 15 of 15 oysters at day 10. The cepB gene was detected by PCR in 12 of 15 oysters at day 4 and in 14 of 15 oysters at day 10. No oysters from the control group were positive for either gene at days 4 or lO. Green fluorescence was detected by flow cytometry at significantly higher levels (P < 0.05) in oysters injected with rsGFP than in other oysters at day 4, but not at day 10. This report indicates the ability to introduce DNA into adult eastern oysters with subsequent gene expression. Future work will involve developing these techniques for enhanced disease resistance in oysters.
The eastern oyster, Crassostrea virginica. is the most important cultured oyster species of the Atlantic and Gulf coasts of the United States, Cryopreservation of gametes and larvae of aquatic organisms has increased in importance in recent years. However, studies on the cryopreservation of sperm and larvae of mollusks have focused on the Pacific oyster, Crassostrea gigas. The present study was conducted to improve cryopreservation of sperm and trochophore larvae and to assess fertilizing ability and male-to-male variation of thawed sperm of the eastern oyster. Sperm were diluted in 12 cryoprotectant solutions composed of Hanks' balanced salt solution without calcium and 0, 5, 10, 15, 20, and 25% (v/v) propylene glycol with or without 0.25 M sucrose. Trochophore larvae were suspended in artificial seawater and 10 or 15% propylene glycol (v/v). Sperm or trochophore larvae were placed in 5-mL macrotubes and allowed to equilibrate for 15 min. The macrotubes were cooled in a controlledrate freezer at a rate of 2.5°C per min until reaching a final temperature of -30°C and were plunged into liquid nitrogen. After storage for 2 weeks, the samples were thawed in a water bath at 70°C for 15 s. Overall, for cryopreservation of sperm and larvae, best results were obtained using 10 or 15% propylene glycol, ThawL,d sperm presented significant male-to-male variation in fertilizing ability. Survival of thawed larvae decreased as the concentration ot larvae per macrotube increased. The procedures developed in this study for sperm and larvae are suitable for production of seedstock in commercial oyster hatcheries.
A population of catfish presumptively identified as channel catfish ( lctalurus punctatus) found in Lake Maurepas in southeast Louisiana matures at a small size and early age ( < l 70 mm TL and <2 years) compared to other populations in southern Louisiana (>500 mm TL and 3 to 4 years). In addition, these catfish have a protracted spawning period. The peak spawning time coincides with cultured channel catfish, but a percentage of the population spawns throughout the summer months. These fish are harvested at a small size and support an important but contentious commercial fishery. The small size and early age at sexual maturity have raised several questions: are these channel catfish, a distinct subspecies, or a hybrid between channel catfish and another ictalurid? These reproductive characteristics would prove useful for research, especially if the taxonomic uncertainty could be resolved. In this study, we differentiated these catfish by fin shape and external coloration from all other ictalurids present in Lake Maurepas except for channel catfish and blue catfish (lctalurus furcatus). Blue catfish were differentiated by the absence of spots, anal fin shape and anal fin ray count. Genome size (cellular DNA content) determined for Lake Maurepas catfish in this study (2.11 +/- 0.01 pg; N = 36) agreed with values for the Kansas strain of channel catfish (2.11 ± 0.01 pg; N = 15 ). Staining of chromosomes to determine the location (chromosome pair) of the nucleolar organizing region (NOR) revealed that the NOR for Lake Maurepas catfish did not differ from that of channel catfish. In addition, NOR for hybrids of Lake Maurepas catfish x blue catfish were located on heteromorphic chromosomes. A segment of an immunoglobin gene was sequenced from channel catfish, Lake Maurepas catfish, blue catfish, and black bullhead (lctalurus melas). The nucleotide sequences for channel catfish and Lake Maurepas catfish were identical, while differences were found among the other catfishes. These data indicate the existence of a distinct population of channel catfish in Lake Maurepas that possesses traits useful for research.
The modern environmental advocacy emerged in the 1960s in response to highly visible examples of environmental degradation such as air and water pollution. extinctions of animals and plants, and major ecosystem disruptions caused by logging, mining, dam building and mechanized agriculture. More recently, advocacy has been extended to include global concerns related to the environmental impacts of expanding human populations, widespread poverty, global climate change and unprecedented losses of biodiversity. The worldwide impact of environmental advocacy groups has increased dramatically in the recent past because of Improved methods of communication, widespread media access and a large infusion of financial resources. For 15 years these groups have criticized specific sectors in aquaculture, but within the past 5 years they have begun to actively target aquaculture as a collective activity. The reasons for this shift in criticism range from legitimate identification of potential problems In aquaculture to fund-raising efforts by advocacy groups based on opinion polls of their memberships. Given these current trends, it appears that this criticism will not diminish in the future and has the potential to influence public opinion, consumer behaviour, and the activities of policymakers and regulatory agencies. Given these realities, it is incumbent upon those involved in aquaculture to gain an appreciation of the importance of responding sensibly to criticism. This includes developing an understanding of the relevant issues, communicating effectively with and through popular media, becoming familiar with the goals and tactics of advocacy groups, and increasing involvement in the formulation of policies and regulations as Individuals and representatives of groups.
Perkinsus marinus infection intensity in Crassostrea virginica can be quantified without killing of oysters by determining parasite density in hemolymph samples incubated in fluid thioglycollate medium (FTM). The goal of this study was to improve existing protocols for counting of P. marinus in oyster hemolymph. Specifically, the objectives were to examine the effects on parasite number and diameter of: 1) adding supplements to FTM such as lipid and oyster extract; 2) incubating with various FTM preparations with and without agar or beef extract; 3) incubating with various hemocyte densities (10^5, 10^6, and 10^7 hemocytes/mL of rTM) in a constant FTM volume; 4) incubating with different volumes of FTM (0.2 mL, 1.0 mL. 5.0 mL, and 25.0 mL); and 5) sodium hydroxide digestion of cellular debris. From these results, an improved hemolymph protocol was developed. The diameters and numbers of enlarged parasites or hypnospores in hemolymph of 20 oysters measured by the improved protocol and the standard FTM hemolymph assay of Gauthier and Fisher were compared. Finally, the standard and improved protocols were compared with the FTM body burden assay. The diameter of hypnospores from samples processed with the improved protocol (26 ± 13 11,m) was significantly greater than the diameters from samples processed with the standard protocol ( 10 ± 4 µm). The number of hypnospores in samples processed with the improved protocol (8.6 x 10^3 ± 3.3 x 10^3) was significantly greater than the numbers in samples processed with the standard protocol (l.9 x 103 ± 3.4 x 10.1)_ Results of the body burden assay were significantly correlated with results of the standard hemolymph assay and with results of the improved hemolymph assay. The coefficient of determination (r^2 = 0.7602) and slope (0.91189) of the regression of the FTM body burden assay against the improved FTM hemolymph assay was improved from the coefficient of determination (0.5543) and slope (0.61257) of the regression of the FTM body burden assay against of the standard FTM hemolymph assay.
The duplication of genes and entire genomes are believed to be important mechanisms underlying morphological variation and functional innovation in the evolution of life and especially for the broad diversity observed in the speciation of fishes. How did these fish species and their genetic diversity arise? The occurrence of three rounds of genome duplication during vertebrate evolution might explain why many gene families are typically about half the size in land vertebrates as they are in fishes. However, mechanisms of diversity in fish lineages need to be further explained. Here we propose that differential genome duplication of from two to six rounds occurred in different fish lines, offering new opportunities during the radiation of fish lineages. This model provides a fundamental basis for the understanding of their speciation, diversity and evolution.
Factors such as osmotic pressure, extender solution, addition of caffeine, and pH have been shown to affect sperm motility in aquatic species. We evaluated the effects or 18 osmotic pressures, two extender solutions, seven caffeine concentrations, am! a pH range of 3 10 14 on motility of sperm from tetraploid Pacific oysters, Crassostrea gigas. Molility was highest at 1000 mOsmol/kg (mean± SD: 83 ± 14%). Calcium-free Hanks' balanced sail solution yielded significantly higher sperm motility than did artificial seawater. Sperm motility increased with caffeine. concentrations to 20 mM (81 ± 12%) and decreased when concentrations were higher than 50 rnM (55 ± 20% ). Highest motility was obtained at a pH range of from 4 to 12; values outside this range yielded no motility. Addition of 1O mM caffeine to the different pH treatments also enhanced motility significantly. Overall, calcium-free Hanks' balanced salt solution at 1,000 mOsmol/kg, the addition of IO mM caffeine, and a pH of around 10 could be used to enhance sperm motility of tetraplnid Pacific oysters. Our findings would assist the use or motility assays to evaluate the effectiveness of various rel'rigeration or cryopreservatiou procedures. especially outside of the peak spawning season, when sperm motility can be low and variable.
The eggs of ornamental (koi) common carp Cyprinus carpio were stored at ambient temperature (-22-25 C) and at refrigerated temperatures (0-20 C) in extenders at different osmolalities. The treatment<; evaluated were dry (control), calcium-free Hanks' balanced salt solution (C-F HBSS), salt (NaCl), synthetic ovarian fluid (SOF), and Kurokura #2 (K2). In the first study, eggs were placed in extenders at osmolalities ranging from 130 to 450 mOsmol/kg and were fertilized after 2 h. The percentage of eyed embryos (our measure of fertilization capacity) was calculated 24 h later, and percent hatching was calculated at 60 h. Fertilization capacity of eggs suspended in CF HBSS (28%) or SOF (37%) was highest (P = 0.0001) at 250 mOsmol/kg, while eggs stored dry (control) had a fertilization capacity of 24%. Fertilization capacity of eggs suspended in NaCl (40%) or K2 (39%) was highest (P = 0.0001) at 200 mOsmol/kg. The percent of eyed embryos and percent hatch were found to be positively correlated (r = 0.9914). In the second study, eggs were stored in these extenders with the most effective osmolality from the previous study to evaluate percent eyed embryos and hatching over time. Samples of eggs were fertilized at every hour for 7 h. Eggs in the extenders C-F HBSS and SOF yielded the highest (P = 0.0001) percent eyed embryos during 7 h. Percent hatch of these eggs was not significantly different (P = 0.1258) among treatments at each time interval. Eggs stored in the extenders SOF, C-F HBSS, and NaCl had higher fertilization capacity (P = 0.0271) at 7 h than did the dry control. Eggs were also stored at refrigerated temperatures in these four extenders at the most effective osmolalities from the first study. A dry control (no extender) was also compared. The third study compared quality of eggs stored for 0, 2, 4, or 6 h in each of the extenders at 5 C or at ambient temperature (-22- 25 C). Eggs suspended in C-F HBSS had significantly higher fertilization capacity at ambient temperature over time than did eggs stored in NaCl, SOF, K2, or the dry control. Eggs suspended in C-F HBSS and the dry control had significantly higher fertilization capacity at 5 C over time than did eggs stored in NaCl, SOF, or K2. Eggs held dry had higher hatch at ambient temperatures (P = 0.0001) and at 5 C (P = 0.0002) over time than did eggs stored in any extender. At 6 h, fertilization capacity with eggs in C-F HBSS or K2 was higher than with NaCl, SOF, or the dry control. The fourth study used C-F HBSS (250 mOsmol/kg) as the extender to evaluate fertilizing and hatching ability during storage at temperatures 0, 5, lO, 15, 20, or 25 C. Eggs were fertilized after 0, l, 3, 6, 9, or 12 h of storage. Eggs stored at 15 C bad significantly higher fertilization capacity (P = 0.0001) than at any other temperature. Eggs stored at 15 C and 10 C had significantly higher hatch (P = 0.0001) than at any other temperature. Fertilization capacity at 12 h was significantly higher in eggs stored at 10 C (33%) or 15 C (29%) than at any other temperature. Storage of koi carp eggs in C-F HBSS at refrigerated temperatures extended fertilizing ability for as long as 12 h compared to storage in NaCl, SOF, K2, or the dry control.
The mammalian sex determining gene, SRY, is the founding member of the new growing family of Sox (SRY-like HMO-box gene) genes, Sox genes encode transcription factors with diverse roles in development, and a few of them are involved in sex determination and differentiation. We report here the existence of Sox genes in the rice field eel, Monopterus albus, and DNA sequence information of the HMG box region of five Sox genes. The Sox 1, Sox4 and Sox14 genes do not have introns in the HMO box region. The Sox9 gene and Sox17 gene, which each have an intron in the conserved region, show strong identity at the amino acid level with the corresponding genes of mammals and chickens. Similar structure and identity of the Sox9 and Sox17 genes among mammals, chickens and fish suggest that these genes have evolutionarily conserved roles, potentially including sex determination and differentiation.
A control system was designed to raise and maintain water temperatures within ~ 0.03-ha earthen ponds to a range conducive for spawning (24 -30 "C) channel catfish lta!urus punctatus. Heating was done during February 2001 to April 2001, when temperatures would have otherwise prohibited spawning ( < 24 'C). Temperature was increased from ~ 1O °C (ambient) by ~ 2 ·c per day, and maintained at ~ 27 'C, by the addition of geothermally warmed water ( ~ 36 ')C). The control system substantially increased the controllability and precision of heating ponds compared to manual operation. Systems were designed to control sets of four ponds. In designing this control system, consideration of biological constraints was essential. Reproduction in channel catfish is most strongly influenced by temperature. Because cold fronts are common during the winter and early spring (January- March), it was essential to ensure that pond temperatures did not fall below the range for spawning. Constraints on the heating rate and temperature variability to maintain fish health and stimulate spawning behavior were considered. Components of the control system included temperature measurement devices (type-T thermocouples), a central electronic control unit, electronic switches and electrically actuated ball valves. In response to the temperature sensed by each thermocouple,the controller sent a message to close or open tile valve. When the valve was opened, warm water was added to the pond to increase the average pond temperature. Hardware and algorithm design and initial system testing were the major components of this project. The final design incorporated information on relevant biological parameters and safety features including peak pond temperature, independent aeration and water pressure control mechanisms. Initial results indicate successful control of this biological system, and ongoing studies suggest similar mechanisms may be used for additional control objectives. In particular, this system could be used to vary pond temperatures to study biological responses and to cool ponds by addition of well water during summer months.
The spawning of channel catfish Ictalurus punctatus in earthen ponds is limited to the spring and early summer (late April through June) in North America, when water temperatures range from 24°C to 30°C. Temperature appears to be the primary factor that influences the timing of spawning in channel catfish. The heating of ponds will allow the spawning season to begin early. During the late winter and spring (February to early May) of 1997, 1999, 2000, and 2001. 0.03-ha earthen ponds containing male and female channel catfish broodstock ( 1.3-4.1 kg, 440- 680 mm total length) were heated at about 2°C/d by adding geothermally heated water (36°C) and then maintained within the range of 24-30°C. Four spawning containers were added to each pond and checked for eggs at 3-d intervals. Eggs were removed and evaluated for fertilization percentage. Over the 4 years, spawning in heated ponds occurred from 20 to 62 din advance of spawning in unheated (control) ponds, and the period of egg production possible within one season was doubled. Spawning usually commenced within 1-2 weeks of the observation of nesting activity, and 2-4 weeks were required to collect a total of four masses ( our criterion for the full onset of egg production in a heated pond). Fertilization was 87% +/- 8% (mean +/- SD) in spawns collected before the onset of spawning in unheated ponds (N = 67 masses) and 87% +/- 5% in spawns collected during the normal season (N 26 masses). There were no significant differences in the weight (P = 0.08) and fertilization percentage (P 0.88) of egg masses collected early and during the normal spawning season. Problems encountered included disease outbreaks of Cleidodiscus sp., Jchthyophthirius multifilis, and Flexibacter columnaris in broodstock. Future studies should evaluate methods to reduce the added costs of heating ponds and incubating egg masses.
In chickens, the difference in DNA content between the Z and W sex chromosomes is measurable as a 2% greater amount of DNA in cells of males (ZZ) vs. females (ZW). High-resolution measurement of cellular DNA content (genome size) is possible by use of flow cytometry. In this technique, nuclei are stained with a nucleic-acid specific fluorochrome, and are passed single-file through a high-intensity light source such as a laser beam. The fluorescence emitted from each nucleus is directly proportional to the amount of DNA present, and can be used to quantify genome size on a per-cell basis. This technique is rapid (two or three samples per minute after a 10-minute staining period), accurate, and straightforward. Commercially available clinical and research instruments are generally expensive (US$80,000 to 400,000), but it may be possible to couple the principles of flow cytometry with existing egg handling equipment to enable rapid throughput for identification of sex in chicks before hatching.
This paper addresses four types of biosecurity problems that result in varying degrees of uniqueness from cryopreservation of material from aquatic species: (a) disease transmission, (b) introduction of exotic species, ( c) genetic consequences for target species, and ( d) genetic consequences for ecosystems. Cryopreservation has been studied in aquatic species since the 1950s. Interest in frozen storage of gametes and early life stages of aquatic organisms has expanded recently and advances have made the technology available for commercial application. Integration of cryopreservation into commercial aquaculture has begun and could expand rapidly in response to global demands for improved broodstock and consistent production of seedstock. Frozen bull semen represents a multi-billion dollar global industry that provides a clear example of how a corresponding industry could develop for aquaculture species. This is because it is much simpler and cheaper to ship straws containing millions of frozen gametes or larvae than it is to ship even a few broodstock animals. These, and other benefits of cryopreservation, offer advantages for development, maintenance, and distribution of improved lines, but they also present a biosecurity challenge to guard against transfer of pathogens and other problems through space and time. Diseases of today could be disseminated rapidly throughout the world, and diseases of yesterday could re-emerge in the future through frozen samples. The potential for disease transfer could eventually present a stumbling block to the responsible development of this valuable technology for aquaculture. It is also possible that a lack of forethought will lead to unnecessary transfer of diseases or nonnative species with cryopreserved samples before proper awareness can be developed. The lack of specific guidelines and regulations, and the rapid expansion of cryopreservation research and application intensify this problem. Fortunately, regulatory frameworks, treaties, and agreements addressing the transfer of aquatic animals and aquaculture products exist through organizations such as the United States Department of Agriculture, the United States Fish and Wildlife Service, and the Office International des Epizooties, the world organization for animal health that works to harmonize transfer regulations in over 150 member countries. Such mechanisms can be updated to include oversight of cryopreserved materials, especially with concern for the risks of temporal transfer of organisms ( e.g., years or decades after collection), and the expanded opportunities for transfer provided by cryopreservation ( e.g., sperm samples that would otherwise only survive for a few days). Existing procedures are also available to eliminate or minimize contamination of aquaculture broodstock, gametes, and early life stages. Responsible development of biosecurity programs for cryopreservation in aquatic species can occur before significant commercial application takes place. This effort would be assisted greatly by transferring successful practices from the existing cryopreservation industries established for livestock.
The commercial-scale production of fish by use of artificial (induced) spawning would require reliable, large-volume sources of sperm. Cryopreservation can be used to preserve and store sperm within commercial and research germplasm repositories, but is limited in its application to aquaculture. Straw volume and cooling chamber size restrict the quantity of sperm that can be frozen. and straws must be filled by hand. In contrast, the dairy industry has refined methods for freezing of bull sperm, including automation of straw filling and the use of large cooling chambers. These methods could be used for commercial-scale cryopreservation of fish sperm, although application would require testing. To supply sperm in large volumes, bags originally developed for swine semen could be cooled using dairy protocols and used as a container for fish sperm. The current study documented the use or commercial-scale dairy cryopreservation techniques for the production of hybrids of channel catfish Ictalrus punctatus (female) by blue catfish Ictalurus furcatus. Four cryoprotectants (methanol, dimethyl sulfoxide, dimethyl acetamide, and glycerol) were initially evaluated for use with blue catli,h sperm. During May 2000 and March to April 2001, suspensions of blue catfish sperm were cryopreserved with 10% methanol in 0.5-mL French straws and in commercial swine semen bags (Cochette bags, IMV International, Minneapolis, Minnesota. USA). Cryopreservation took place at a dairy breeding cooperative, using technology employed for bull semen. Sperm motility before freezing was 26 :t: 18% during Year 1 (2000) and 62 :t: 30% during 2001. Sperm were thawed at 40 C and used to fertilize the eggs of channel catfish (yielding hybrids). Motility after thawing for sperm rrn.1.en in 0.5-mL straws was 11 +/- 10% during 2000 and 50 +/- 24% during 2001. Motility after thawing was 41 +/- 17% for sperm frozen in swine semen bags in 5-mL aliquots and 43 +/- 10% for sperm frozen in 10-mL aliquots. Neurulation of eggs fertilized with thawed sperm from straws was in ± 13% during 2000 and 54 +/- 27% during 2001. Neurulation was 57 +/- 24% using sperm frozen in swine semen bags in 5-m L aliquots and 55 +/- 10% using sperm frozen in 10-mL aliquots. There was 110 correlation between sperm motility before freezing (in 0.5-mL straws) and after thawing during 2000 (r = 0.52) or during 2001 (r = 0.49). In addition, there was no correlation between initial motility and neurulation of channel catfish eggs fertilized using thawed sperm during 2000 (r = 0.11) or during 2001 (r = 0.29). Sperm of blue catfish can thus be cryoprescrved at a commercial scale using dairy protocols and can be made available for the production of hybrid catfish when viable eggs are available.
The Sox9 gene encodes a transcription factor that is critical for testis determination and chondrogenesis in vertebrates. Mutations in human SOX9 cause campomelic dysplasia, a dominant skeletal dysmorphology syndrome often associated with male to female sex reversal. Here we show that the Sox9a gene was duplicated during evolution of the rice field eel, Monopterus albus, a freshwater fish which undergoes natural sex reversal from female to male during its life, and has a haploid genome size (0.6- 0.8 pg) that is among the smallest of the vertebrates. The duplicated copies of the gene (named Sox9al and Sox9a2) fit within the Sox9 clade of vertebrates, especially in the Sox9a subfamily, not in the Sox9b subfamily. They have similar structures as revealed by both genomic and cDNA analysis. Furthermore, both Sox9al and Sox9a2 are expressed in testis, ovary, and ovotestis; and specifically in the outer layer (mainly gonocytes) of gonadal epithelium with bipotential capacity to form testis or ovary, suggesting that they have similar roles in gonadal differentiation during sex reversal in this species. fhe closely related gene structure and expression patterns of the two sox9a genes in the rice field eel also suggest that tl1ey arose in recent gene duplication events during evolution of this fish lineage.
We developed methods for the cryopreservation of sperm of the endangered Colorado pikeminnow Ptychocheilus lucius. Sperm were collected from a captive broodstock population of Colorado pikeminnow reared and maintained at the Dexter National Fish Hatchery and Technology Center. Our objectives were to (I) evaluate the effects on sperm motility of 24-h storage in Hanks' balanced salt solution (HBSS); (2) characterize sperm motility and duration; (3) examine the relationship between sperm motility and osmotic pressure; (4) examine the effect of four cryoprotectants (dimethyl sulroxide [DMSO], dimethyl acetamide [DMA], glycerol, and methanol [MeOH] at two concentrations I 59fJ and I 0% I) on postthaw motility; and (5) compare the effect of two cooling rates (40°C/min and 4°C/min) on postthaw motility. The sperm samples diluted with HBSS retained higher motility (mean +/- SD, 77 +/- 22%; n = 9) than did undiluted samples ( 12 +/- 30%; n = 9) after 24 h of storage. When exposed to HBSS at 274 mosmols/kg or more, few sperm became motile ( --1 % ). Exposure to H BSS at 265 mosmols/kg elicited threshold activation (defined as 10%, motility), and maximum motility (>95%) was observed at 93 mosmols/ kg. The maximum motility of sperm was observed within 10 s after activation with deionized water, and sperm remained motile for 57 s. The sperm that were cooled al a rate of 40°C/min and cryopreserved with S'fo MeOH retained higher postthaw motility (56 +/- 13%) than did sperm cryopreserved with DMSO, DMA, or glycerol (at 5% and 10%). When the sperm samples were cooled at a rate of 4°C/min, sperm cryopreserved with MeOH (5% or 10%) or DMSO (5% or 10%) retained the highest postthaw motilities(<= 14%). The use of cryopreserved sperm can assist hatchery managers in the production of fish, provide for the long-term conservation of genetic resources, and assist in the recovery or endangered species such as the Colorado pikeminnow.
Sperm were collected in Florida from wild common snook, Centropomus undecimalis (Bloch). and were shipped to Louisiana State University for analysis and cryopreservation, Threshold activation of sperm (10% motility) occurred at 370 mOsmol kg 1. and complete activation occurred at 680 mOsmol kg^-1. These values were significantly different. Sperm samples stored at 1 °C in Hanks' balanced salt solution (HBSS) or in 0.6% NaCl solution at 200 mOsmol kg- 1 retained motility for as long as 22 days. Mean motility remained above 50% for 9 days for sperm stored in HBSS and for 7 days for sperm stored in NaCl solution. Sperm exposed to 5% dimethyl acetamide (62 ± 10%; mean ± SD), 10% dimethyl sulphoxide (DMSO) (39 ± 16%). 5% glycerol (26 ± 5%) or 10% glycerol (6 ± 2 % ) for 30 min had significantly lower motility than did unexposed sperm (89 ± 9% ). When used as a cryoprotectant, samples frozen with 5% or 10% DMSO or 5% methanol had significantly higher post-thaw motility than did samples frozen with other cryoprotectants. Sperm cryopreserved with 10% DMSO (38 ± 12%) had significantly higher post-thaw motility than did sperm cryopreserved with 15% DMSO (19 ± 10%) or 20% DMSO (4 ± 4%). There were no significant differences in hatch rates of eggs fertilized with fresh sperm (54 ± 29%) or cryopreserved sperm (41 ± 3.5%). Survival to first feeding was not different between fish produced with fresh sperm (37 ± 30%; range, 0-86%) or with thawed sperm (24 ± 29%: 0-77%). Transport of sperm to a cryopreservation laboratory and back to a hatchery for thawing and use enabled collaboration between groups with specific expertise and provides a model for the application of cryopreservation by transport of fresh and frozen samples.
Swordtails and platyfish of the genus Xiphophorus are valuable models for biomedical research and are also commercially raised as ornamental fish valued by aquarists. While research use and commercial interest increases yearly in these fish, cryopreservation of sperm is unexplored in this genus. Xiphophorus are live-bearing fishes characterized by small body sizes, limited sperm volumes, and internal fertilization, an atypical reproductive mode for fish. These attributes make research involving cryopreservation of Xiphophorus germplasm challenging. To explore methods for sperm cryopreservation, this study evaluated the effect of different loading volumes of sperm suspension in 0.25-ml French straws, different dilution ratios of sperm to extender, an osmolality range of extender without cryoprotectant and with dimethyl sulfoxide (DMSO) as cryoprotectant, and short-term storage at room temperature and 4 8C after thawing. No significant difference in sperm motility due to straw loading volume was observed after thawing. Sperm motility was observed to decrease with increasing dilution. The osmolality of Hanks’ balanced salt solution (HBSS) without cryoprotectant in which the highest sperm motility (67%) was observed was 320 +/- 3 mOsm/kg, which was also the osmolality of X. helleri blood plasma. When cryopreserved with 10% DMSO, however, the highest motilities within 10 min after thawing were observed with HBSS in the range of 240–300 mOsm/kg. Sperm suspended in HBSS at 320 mOsm/kg with a dilution factor of 100 maintained motility for 24 h at room temperature, but persisted for 10 days when stored at 4 8C. These results provided the first evidence that cryopreservation may be applied to conservation of genetic resources in live-bearing fishes.
In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas. This represents the first application of the DSC technique to sperm cells from nonmammalian species. Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20°(/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA). Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment "ball-on-stick" model with a "ball" 1.66 µm in diameter and a "stick" 41 µm in length and 0.14 µm wide. Similarly, sperm cells of tetraploid oysters were modeled with a "ball" 2.14 µm in diameter and a "stick" 53 µm in length and 0.17 µm wide. Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, Vb, of 0.6 V0, where V0 is the isotonic (or initial) cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (LP8 and E,p) were determined. The combined-best-fit membrane permeability parameters at 5 and 20°C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: LP8 = 0.30 x 10-15 m3/Ns (0.001 7 µm/min-atm) and Etp = 41.0 kJ/mole (9.8 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: LP8[cpa] = 0.27 x 10-15 m3/Ns (0.0015 µm/min-atm) and E,p[cpa] = 38.0 kJ/mole (9.1 kcal/mole). Similarly, the combined-best-fit membrane permeability parameters at 5 and 20°C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: LP == 0.34 x 10- 15 m3/Ns (0.0019 µm/min-atm) and E,P = 29.7 kJ/mole (7.1 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: LP8[cpa) = 0.34 x 10-15 m3/Ns (0.0019 µm/min-atm) and E, [cpa] = 37.6 kl/mole (9.0 kcal/mole). The parameters obtained P in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70 'C/min. These theoretical cooling rates are in close conformity with empirically determined optimal rates of colling sperm cells from Pacific oysters, C. gigas.
Interest in the culture of snappers (family Lutjanidae) has developed throughout the world because of declines in wild stocks combined with a consistent high demand and market value. Some snappers, such as the red snapper Lutjanus campechanus have proven to be difficult to spawn and culture in captivity. As part of a larger study to improve propagation techniques for red snapper, procedures were developed for the collection, handling, and commercial-scale cryopreservation of sperm. Utilization of cryopreserved sperm in spawning of red snapper allows efforts to be focused on maintaining female broodstock, monitoring ovarian development, and increasing efficiency during the strip-spawning process. Red snapper were collected during the 2000 and 2001 spawning seasons (May to August) off coastal Louisiana by hook and line. Testes were surgically removed and sliced to release sperm. Sperm were collected in 50-ml centrifuge tubes and diluted 1:3 (v:v) with calcium-free Hanks’ balanced salt solution. Dimethyl acetamide, dimethyl sulfoxide, methanol, and glycerol were evaluated as cryoprotectants. Dimethyl sulfoxide produced the smallest reduction in motility of sperm cells in acute toxicity trials and produced the highest motilities after thawing (71 F 16%) in initial cryopreservation trials. During the 2-year study, sperm samples from 20 red snapper were frozen using commercial-scale cryopreservation methods developed for dairy bulls. Although sperm motility after thawing was typically greater than 80%, comparison with fresh samples revealed a significant difference in motility ( P = 0.048). Refrigerated and cryopreserved sperm samples with motilities above 80% were used in fertilization trials. Fertilization rates were variable among females (11–85%), although results with refrigerated (non-frozen) and cryopreserved sperm were highly orrelated (r = 0.85). These results demonstrate that refrigerated and cryopreserved sperm were each effective for the artificial fertilization of red snapper eggs.
The objectives of this study were to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio, as well as somatic relationships of body length, body weight, and testis weight to sperm density in the platyfish Xiphophorus couchianus. Sperm motility and survival duration after thawing were significantly different between cryopreservation with dimethyl sulfoxide (DMSO) and glycerol, with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as cryoprotectant, the highest motility after thawing was observed with Hanks’ balanced salt solution (HBSS) across a range of 240–300 mOsm/kg. Samples cooled from 5 to -80 8Cat 258C/min yielded the highest post-thaw motility, although no significant difference was found for cooling rates across the range of 20–30 8C/min. In addition, the highest motility after thawing was found in samples equilibrated from 10 to 30 min with 14% glycerol and cooled at 25 8C/min. The post-thaw motility declined rapidly with use of 10% glycerol and cooling at 5 8C/min across the equilibration range of 10 min to 2 h. Sperm motility with a dilution ratio of sperm to extender of 1:10 was not different at 10 min after thawing with those samples at greater dilutions, but declined significantly from Day 1 after thawing and showed lower survival duration when stored at 4 8C. However, the additional dilution of sperm solutions with HBSS (300 mOsm/kg) immediately after thawing significantly slowed the decline of motility and prolonged the duration of survival. Based on the above findings, the highest average sperm motility (78 +/- 3%) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsm/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS–glycerol of 1:20, equilibrated for 10 min, cooled at 25 8C/min from 5 to -80 'C before plunging into liquid nitrogen, and thawed at 40 'C in a water bath for 7 s. If diluted within 5 h after thawing, sperm frozen by the above protocol retained continuous motility for 15 days when stored at 4 8C.
Sperm cryopreservation for fishes with internal fertilization is essentially unexplored although many species of these fishes are valuable biomedical research models. To explore methods for sperm cryopreservation within the live-bearing genus Xiphophorus, this study used X. helleri to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio. Sperm motility and survival duration after thawing showed significant differences among different cryoprotectants with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as the cryoprotectant, the highest motility after thawing was observed with Hanks balanced salt solution (HBSS) at 300 mOsmol/kg. Samples cooled from 5 to )80 'C at 20'C/min yielded the highest post-thaw motility although no significant difference was found in the first 4 h after thawing for cooling rates across the range of 20–35 C/min. Evaluation of equilibration time revealed no significant difference between 20 min and 2 h, but the highest motility at 10 min after thawing was found with a 20-min equilibration. Dilution ratios of sperm-to-extender at 1:20, 1:60, and 1:120 showed no significant differences in motility and survival duration after thawing, but the dilution of sperm solutions with HBSS (320 mOsmol/kg) immediately after thawing reduced the decline of sperm motility, and significantly prolonged the survival duration. Based on these findings, the highest average sperm motility (77%) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsmol/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS–glycerol of 1:20, equilibrated for 10 min, cooled at 20 'C/min from 5 to )80 'C before being plunged in liquid nitrogen, and thawed in a 40 C water bath for 7 s. If diluted immediately after thawing, sperm frozen by the protocol above retained continuous motility after thawing for more than 8 days when stored at 4'C.
Cryopreservation of sperm from tetraploid organisms (the possession of four chromosome sets) is essentially unexplored. This is the first cryopreservation study to address sperm from tetraploid Pacific oysters, Crassostrea gigas, and addresses the commercial production of triploid oysters (three chromosome sets). Initial motility, refrigerated storage of undiluted sperm, osmolality of extender solutions, sperm concentrations, equilibration time, and cryoprotectants of propylene glycol and dimethyl sulfoxide were evaluated with sperm from diploid and tetraploid oysters. Unlike most teleost fishes, in which the duration of active motility is typically brief, the motility of sperm from oysters lasts for hours. The present study showed that responses to treatment effects by sperm from tetraploids were different from diploids. The majority of tetraploid experiments resulted in less than 10% motility after thawing and less than 5% fertilization. The highest fertilization obtained for thawed sperm was 96% for sperm from diploid oysters and 28% for sperm from tetraploid oysters. Differential responses to treatments by sperm from tetraploid and diploid oysters may be due to differences in gonadal development. However, the use of cryopreserved sperm from tetraploid Pacific oysters produced 100% triploid offspring by fertilization of eggs from diploid females as determined by flow cytometry of larvae. This study demonstrates that sperm from tetraploid oysters can be collected, frozen, and stored for production of triploid offspring.
To provide necessary standardization of procedures for cryopreservation of sperm, a spectrophotomeric method was developed to determine the sperm concentration of diploid and tetraploid Pacific oysters, Crassostrea gigas.Wavelengths of 380,550,581 and 780 nm were compared, and 550 and 581nm were found to be the most sensitive and reliable. A linear relationship between sperm concentration and photo-metric absorbance was observed for sperm concentrations between 2 * 10^7 and 2 * 10^9 cells mL^-1. The regression equation for the standard curve at 550 nm for sperm of diploid oysters was Y = -8.528 + 1.165 log X. The equation for sperm of tetraploid oysters was Y = -8.844 + 1.236 log X. The equation at 581nm for sperm of diploid oysters was Y = -8.07 + 1.104 log X. The equation at 581nm for sperm of tetraploid oysters was Y = -8.3311 + 1.167 log X. Comparisons derived from the standard curves at 581nm between observed values and the predicted values indicated good agreement for sperm from diploid (coefficient of determination, r^2 50.983) and tetraploid (r2 50.980) oysters.
The purpose of this study was to extend previous work on the cryopreservation of channel catfish (Ictalurus punctatus) sperm. The objectives were to compare the effects of freezing and thawing on motility of sperm for: (1) 1 or 48-h exposure before freezing to 5% methanol and use of 0.5 or 0.25 mL straws; (2) 1 h or 5-day exposure before freezing to 5% methanol; (3) cooling at 45 or 3 8C/min; (4) thawing at 30, 40 or 50 8C using 5 or 10 s duration, and (5) cryopreservation with 5 or 10% methanol of samples from 50 males to analyze male-to-male variation. No differences were found in motility reduction for 1 or 48 h exposure times in 5% methanol, for use of 0.5 or 0.25 mL straws, or for 1 h or 5-day exposures in 5% methanol. A cooling rate of 45 8C/min resulted in lower motility reduction (33 +/- 9%) than a rate of 3 8C/min (83 +/- 13%) (P = 0.002). A thawing temperature of 50 8C resulted in lower motility reduction (25 +/- 14%) than 30 8C (51 +/- 21%) or 40 8C (59 +/- 11%)(P = 0.001). A thawing duration of 10 s resulted in lower motility reduction (38 +/- 12%) than a duration of 5 s (52 +/- 12%) (P = 0.005), and there was an interaction between thawing temperature and duration (P = 0.050). A concentration of 5% methanol resulted in lower motility reduction (43 +/- 17%) than 10% methanol (67 +/- 14%) (P = 0.001). Regression analysis showed no relationship between motility before freezing and after thawing for 5% methanol (r^2 = 0.012) or 10% methanol (r^2 = 0.011).
Sperm cryopreservation of live-bearing fishes, such as those of the genus Xiphophorus is only beginning to be studied, although these fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. To explore optimization of techniques for sperm cryopreservation of these fishes, this study measured the volumetric shrinkage response during freezing of sperm cells of Xiphophorus helleri by use of a shape-independent differential scanning calorimeter (DSC) technique. Volumetric shrinkage during freezing of X. helleri sperm cell suspensions was obtained in the presence of extracellular ice at a cooling rate of 20 8C/min in three different media: (1) Hanks’ balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol; and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of 33.3 mm in length and 0.59 mm in diameter with an osmotically inactive cell volume (Vb) of 0.6Vo, where Vo is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) of the Xiphophorus helleri sperm cell membrane were determined. The best-fit membrane permeability parameters at 20 8C/min in the absence of CPAs were: Lpg = 0.776 * 10^-15 m^3/Ns (0.0046 mm/min atm), and ELp = 50.1 kJ/mol (11.97 kcal/mol) (R^2 = 0.997). The corresponding parameters in the presence of 14% glycerol were Lpg[cpa] = 1.063 * 10^-15 m^3/Ns (0.0063 mm/min atm), and ELp[cpa] = 83.81 kJ/mol (20.04 kcal/mol) (R^2 = 0.997). The parameters in the presence of 10% DMSO were Lpg[cpa] = 1.4 * 10^-15 m^3/Ns (0.0083 mm/min atm), and ELp[cpa] = 90.96 kJ/mol (21.75 kcal/mol) (R^2 = 0.996). Parameters obtained in this study suggested that the optimal rate of cooling for X. helleri sperm cells in the presence of CPAs ranged from 20 to 35 8C/min and were in close agreement with recently published, empirically determined optimal cooling rates.
Gamete preservation techniques are essential in animal husbandry as well as in assisted reproduction for humans. In this research we attempted to use 3 different sperm preservation techniques in combination with newly developed techniques for intracytoplasmic sperm injection (ICSI) to fertilize eggs of a teleost fish, the Nile tilapia (Oreochromis niloticus). Of 47 eggs injected with fresh sperm, 11 (23%) were fertilized, 5 developed abnormally, and 4 developed normally and hatched; from these, one grew to adulthood. Nuclear DNA content of 4 of the abnormal embryos indicated that they were diploid. Flow cytometric analysis of a blood sample from the surviving ICSI fish collected 2 months after fertilization indicated that the fish was diploid. Of 45 eggs injected with cryopreserved sperm, 9 (20%) developed to the blastula stage. Of 40 eggs injected with sperm preserved in 70% methanol, none were fertilized. No injections were possible with freeze-dried Nile tilapia sperm owing to technical difficulties during manipulation. Al-though the findings described here are limited, they provide the first steps toward using sperm preservation methods in addition to cryopreservation for fertilization in fishes.
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 (70F10%) 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.
The coppernose bluegill (CBG) Lepomis macrochirus purpurescens is a bluegill subspecies that is native to Florida. The CBGs and their hybrids are of interest as potential food fish. Our objectives were to (1) develop an osmolality activation curve for CBG sperm, (2) determine suitable cryoprotectants for the cryopreservation of CBG sperm, and (3) demonstrate the ability of cryopreserved CBG sperm to fertilize eggs. Sperm were stripped from mature CBGs (mean +/- SD = 113 +/- 35 g, n = 10), diluted in Hanks’ balanced salt solution (HBSS; osmolality = 300 mosmols/kg), and stored at 4'C. An activation curve was generated by exposing the sperm to solutions prepared by serial dilution of HBSS with de-ionized water. The sperm remained inactive at osmolalities above 265 mosmols/kg. Dilution below 115 mosmols/kg was required for complete activation. The motility of sperm stored in these solutions was estimated at 24-h intervals. Stripped sperm motility (mean +/- SD) declined from 90 +/- 7% initially to 26 +/- 11% after 6 d of storage at 4'C. Motility of sperm from crushed testes declined from 80 +/- 8% at collection to 28 +/- 10% at 1 d of storage. By day 5, motility of sperm from crushed testes was 9 +/- 9%; no motility was observed at 6 d. Sperm were exposed to 10% concentrations of the cryoprotectants dimethyl acetamide (DMA), dimethyl sulfoxide (DMSO), and methanol (MeOH), and motility was evaluated at 15-min intervals for 60 min. Initially, motility was 81 +/- 11% for the control sperm. At 60 min, motility was 0% for DMA, 30 +/- 24% for DMSO, 78 +/- 10% for MeOH, and unchanged for the control. For samples frozen in a controlled-rate freezer (CRF), post-thaw motility was 26 +/- 10% for samples with 10% DMSO and 51 +/- 18% for samples with 10% MeOH. For samples frozen by means of dairy industry technology (DIT), post-thaw motility was 46 +/- 21% for samples with 10% DMSO and 56 +/- 13% for samples with 10% MeOH. Overall, samples frozen in 10% MeOH had significantly higher post-thaw motility (P = 0.043) than did those frozen in 10% DMSO. However, post-thaw motility did not differ between samples frozen by use of the CRF and DIT (P = 0.13). For sperm frozen in the CRF, fertilization was 15% for samples with 10% DMSO and 50% for samples with 10% MeOH. For sperm frozen by means of DIT, fertilization was 50% for samples with 10% DMSO and 75% for samples with 10% MeOH.
Abalone culture. a developing industry in Baja California. Mexico would benefit from genetic improvement and controlled breeding. The use or cryopreserved sperm would allow gernplasm availability. and this study was designed to develop sperm cryopreservation protocols for red abalone Halio1is r11fesce11s. The acute toxic effects of the cryoprotectants dimethyl sulfoxide (DMSO). propylene glycol (PG). and glycerol (GL Y) were assessed after suspending sperm in different concentrations. whereby cryoprotectant treatments of 10% DMSO and 10% GLY equilibrated for 10 min yielded the highest range of motile sperm in preliminary freezing trials and were used for cryopreservation studies. To determine effective cooling rates, three freezing chambers were tested. Replicate samples of sperm from 4 males were placed in 0.5-mL French straws and frozen using a commercial freezing chamber (CFC) used for bull sperm. a programmable rate chamber (PRC). and a manually controlled styrofoam chamber (MCC). For the CFC. the cooling rate was 16°C/min. from 4°C to -140°C. For the PRC and MCC, it was 1'C/min, from -20°C to -30°C. The samples were held at -30°C for 5 min before being plunged into liquid nitrogen (-196°C) for storage. and each sample was thawed in a water bath at 45°C for 8 s. The quality of thawed sperm was determined by estimating percent motility, evaluating membrane integrity using a dual-staining technique and flow cytometry. and estimating fertilization rate. Statistical analyses were performed using 2-way ANOVA where chamber and treatment were the independent variables. Sperm quality parameters were independent. For motilities, a significant interaction was noted between the cryoprotective treatment and the chamber type. whereby motilities for DMSO and GLY were higher (P = 0.0055) using MCC. membrane integrities were significantly lower after using the PRC than the CFC or the MCC (P = 0.0167). The highest post-thaw motility (48 +/- 7%) was found using sperm suspended in 10% glycerol and frozen in the MCC. The highest percent of intact membranes (56 ± 11%) was for sperm suspended in 10% glycerol and frozen in the CFC. The highest fertilization rate (29 + 10%) was with samples frozen with 10% glycerol in the CFC. The use of cryopreserved sperm from red abalone provides an alternative breeding option for culture and the protocols delineated are the first developed for this species.
Spermatozoal ultrastructure of tetraploid Pacific oysters, Crassostrea gigas, was examined by light and electron microscopy and compared to that of diploid Pacific oysters. Ultrastructure of sperm from tetraploids was compositionally similar to diploids except for overall size and the number of mitochondria. Instead of the four mitochondria always found in sperm from diploid oysters, 44% of sperm from tetraploid oysters had four mitochondria, 53% had five, and 3% had six. The linear dimensions of sperm components such as acrosome height and width, sperm head length and width, mitochondrial height, length of the main piece and end piece of flagellum, and total length of flagellum in tetraploids were approximately 1.25 times larger than corresponding measurements in diploids, which corresponds to a doubled volume in sperm of tetraploids compared to that of diploids. Tetraploid males produced half the number of sperm (2.7 +/- 0.5 * 10^10)g^-1 of gonad wet weight compared to diploid males. Despite the increased sperm sizes in tetraploids, the ratios of the sperm head length to head width, and sperm head length to flagellum length were the same for both ploidy levels for fixed and live samples. The implications of these findings on susceptibility of sperm from diploid and tetraploid oysters to damage from cryopreservation are discussed.
In this report we describe the steps used for the production of Nile tilapia by intracytoplasmic sperm injection (ICSI). To assure a constant and reliable source of eggs for ICSI we used a method where female Nile tilapia, Oreochromis niloticus, spawned when held individually in 80-1 tanks (a single-breeding system). This allowed collection of eggs every 28F9 days (mean F S.D.) from 26 spawning events by 7 females that spawned two or more times. It was common to obtain more than 1000 eggs from a single stripping. The second step in this project involved the extension of egg viability to assure sufficient time for the ICSI procedure. This was accomplished by placing the eggs in Hanks’ balanced salt solution after stripping, which extended the period of fertility for at least 3 h after collection. The third step was to minimize chromosomal damage after ICSI by localization of the metaphase plate within eggs. The maternal chromosomes were found to be ~43 Am from the micropyle (injection site) at the moment of fertilization. The final step was to apply these methods to evaluate the ICSI procedure for tilapia. From a total of 113 Nile tilapia eggs injected with fresh sperm, 7 (5%) were fertilized, 5 (4%) developed abnormally to neurula and 1 (1%) developed normally and reached adulthood. These results demonstrated for the first time that the ICSI procedures here described allow fertilization and subsequent development of Nile tilapia eggs into normal larvae, hatching and beyond. This provides opportunities for the study of basic processes involved in fertilization and zygotic development and expands the utility and range of sperm storage methods such as cryopreservation or freeze-drying.
This study reports the subzero water transport characteristics (and empirically determined optimal rates for freezing) of sperm cells of live-bearing fishes of the genus Xiphophorus, specifically those of the southern platyfish Xiphophorus maculatus. These fishes are valuable models for biomedical research and are commercially raised as ornamental fish for use in aquariums. Water transport during freezing of X. maculatus sperm cell suspensions was obtained using a shape-independent differential scanning calorimeter technique in the presence of extracellular ice at a cooling rate of 20'C/min in three different media: (1) Hanks' balanced salt solution (HBSS) without cryoprotective agents (CPAs); (2) HBSS with 14% (v/v) glycerol, and (3) HBSS with 10% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder with a length of 52.35 lm and a diameter of 0.66 lm with an osmotically inactive cell volume (Vb) of 0.6V0, where V0 is the isotonic or initial cell volume. This translates to a surface area, SA to initial water volume, WV ratio of 15.15 lm^-1. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best fit membrane permeability parameters (reference membrane permeability to water at 0'C, Lpg or Lpg [cpa] and the activation energy, ELp or ELp [cpa]) were found to range from: Lpg or Lpg [cpa] = 0.0053–0.0093 lm/min atm; ELp or ELp [cpa] = 9.79–29.00 kcal/mol. By incorporating these membrane permeability parameters in a recently developed generic optimal cooling rate equation (optimal cooling rate, Bopt = 1009.5exp(-0.0546ELp)(Lpg) SA/WV where the units of Bopt are 'C/min, ELp or ELp [cpa] are kcal/mol, Lpg or Lpg [cpa] are lm/min atm and SA/WV are lm^-1), we determined the optimal rates of freezing X. maculatus sperm cells to be 28 C/min (in HBSS), 47 'C/min (in HBSS + 14% glycerol) and 36 'C/min (in HBSS + 10% DMSO). Preliminary empirical experiments suggest that the optimal rate of freezing X.
Despite some 26 published reports addressing oyster sperm cryopreservation, systematic factor optimization is lack-ing, and sperm cryopreservation has not yet found application in aquaculture on a commercial scale. In this study, the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization of shipped sperm samples from diploid oysters. Evaluation of cooling rates revealed an optimal rate of 5 °C/min to -30 °C followed by cooling at 45 °C/min to -80 °C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations suggested that a low concentration (2%) of polyethylene glycol (FW 200) was effective in retaining post-thaw motility and fertilizing capability when combined with permeating cryoprotetcants such as dimethyl sulfoxide (DMSO), methanol (MeOH), and propylene glycol (P-glycol). However, polyethylene glycol alone was not as effective as MeOH, DMSO, and P-glycol when using the same methods. The highest post-thaw motility (70%) and percent fertilization (98%) were obtained for samples cryopreserved with 6% MeOH. However, this does not exclude other cryoprotectants such as DMSO or P-glycol identified as effective agents in other studies. There was no significant difference in post-thaw motility between straw sizes of 0.25- and 0.5-ml. Equilibration time (exposure to cryoprotectant) of 60 min could be beneficial when the cryoprotectant concentration is low and solution is added in a step-wise fashion at low temperature. Differences in post-thaw sperm quality (e.g., motility or percent fertilization) among individual males were evident in this research. As a consequence, a generalized classification describing males with different tolerances (broad, intermediate, and narrow) to cryopreservation was developed. This classification could be applied to strain or species differences in tolerances to the cryopreservation process. The present study demonstrated that oyster sperm could be collected and shipped chilled to another facility for cryopreservation, and that it could be shipped back to the hatchery for fertilization performed at a production scale yielding live larvae with >90% fertilization. Given the existence of facilities for commercial-scale cryopreservation of dairy bull sperm, the methods developed in the present study for oysters provide a template for the potential commercialization of cryopreserved sperm in aquatic species.
In this study, refrigerated storage and cryopreservation of sperm from the green swordtail Xiphophorus helleri were investigated. Previous cryopreservation research in this species utilized motile sperm because unlike in most Wsh species, Xiphophorus sperm can remain continuously motile after collection for a week with refrigerated storage. However, this species reproduces by internal fertilization, and given the significant requirements for motility within the female reproductive tract and potential limitations on sperm energetic capacities, immobilization of sperm prior to insemination could be used to improve fertilization success. Thus, the goal in this study was to use osmotic pressure to inhibit the motility of sperm after collection from X. helleri, and to test the effect of immobilization on refrigerated storage and cryopreservation. The objectives were to: (1) estimate the motility of sperm at different osmotic pressures, and determine an osmotic pressure suit-able for immobilization; (2) cryopreserve the immobilized sperm, and estimate the motility after thawing with or without dilution, and (3) compare motility of non-immobilized and immobilized sperm after thawing, centrifugation, and washing to remove cryoprotectant. Motility was determined when sperm were suspended in 11 different osmotic pressures (24–500 mOsmol/kg) of Hanks’ balanced salt solution (HBSS). Motility was observed between 116 and 425 mOsmol/kg. Sperm were not motile when the osmolality was lower than 116 or higher than 425 mOsmol/kg. Motility of the immobilized (non-motile) sperm could be activated by changing the osmotic pressure to 291–316 mOsmol/kg, and motility of immobilized sperm from hypertonic HBSS (7425 mOsmol/kg) was significantly higher than that from hypotonic HBSS (6145 mOsmol/kg) after 48 h of storage. At an osmolality of 500 mOsmol/kg, HBSS was used as extender to maintain immobilized sperm during cryopreservation with glycerol as the cryoprotectant. High motility (»55%) was obtained in sperm after thawing when cryopreserved with 10–15% glycerol, and dilution of thawed sperm in fresh HBSS (1:4; V:V) was found to decrease the motility significantly. No difference was found in the motility of thawed sperm cryopreserved with 14% glycerol and extended in 310 and 500 mOsmol/kg HBSS. Washing by centrifugation prolonged the motility of thawed sperm from 24 to 72 h in HBSS at 310 and 500 mOsmol/kg. This study showed that sperm from X. helleri could be immobilized by use of specific osmotic pressures, and that the immobilization did not affect sperm motility after thawing. The immobilization of sperm by osmotic pressure could minimize reduction of the energetic capacities necessary for insemination, traversal, and residence within the female reproductive tract, and fertilization.
A pond heat and temperature regulation (PHATR) model was designed to: (1) predict the temperature for earthen outdoor aquaculture ponds and (2) determine the size of energy transfer mechanisms affecting energy gains or losses for these ponds. The model solves a first order, no-linear differential equation using a 4th order Runge-Kutta numerical method and various input data (weather data, pond characteristics and flow rate data). Output data (predicted pond temperature) was compared to measured pond temperature collected from the warmwater ponds at the Louisiana State University Agricultural Center Aquaculture Research Station, Baton Rouge, Louisiana. The model over-predicted the temperature for unheated ponds by 0.7 'C and for heated ponds by 2.6 'C. Fluctuations in flowrates of warm water used to heat the pond are believed to be responsible for the greater error in predicting heated pond temperatures. On average, the two most important energy vectors for unheated ponds were longwave pond radiation (39%) and longwave sky radiation (31%). At certain times, solar radiation accounted for as much as 49% of all energy transferred to unheated ponds. For heated ponds, on average, important energy transfer mechanisms were longwave pond radiation (25%), longwave sky radiation (19%), warm geothermal-well water (19%) and discharged water (15%). At certain times, solar radiation accounted for as much as 50% and warm well water 60% of all energy transferred to heated ponds.
stract The PHATR model was designed to determine: (1) the temperature and (2) the size of the energy transfer mechanisms for research-sized (400 m^3) outdoor aquaculture ponds in Louisiana. Because various environmental parameters affect the model’s output, a sensitivity analysis was used to identify which factors had a greater affect on the pond temperature. Four environmental parameters (air temperature, solar radiation, wind speed and flow rate of water used to control the pond temperature) were varied one at a time. The temperature for 1 m^3 ponds was modeled for 48 h. The output (pond temperature) for each trial model run was compared to the output of a standard model run. The model’s sensitivity to air temperature varied linearly (r = 0.999) from 0.10 to 0.35 'C/'C. The model’s sensitivity to solar radiation ranged from 0.04 to 0.14 'C/W. The model’s sensitivity to wind speed (dependent on wind speed) ranged from -0.003 to -1.64 'C/(m/s). The model’s sensitivity to the flow of warm water (dependent on the flow of warm water) ranged from 158,074 to 620,845 'C/(m^3/s)/m^3. The model’s sensitivity to the flow of cool water (dependent on the flow of cool water) ranged from 46,375 to 844,873 8C/(m^3/s)/m^3. For all cases, time was found to have an effect on the model’s sensitivity. Ultimately, this data is useful for the design and management of outdoor earthen aquaculture ponds in the Southeastern United States or similar areas.
Spermatozoa from diploid and tetraploid Pacific oysters (Crassostrea gigas) were examined after anisotonic fixation. Morphological anomalies, such as membrane rupture, detached tails, and the formation of tail vesicles (typically associated with damage attributable to procedures such as cryopreservation) were observed; the Mantel-Haenszel Chi-square test indicated a strong association be-tween the anomalies and fixative osmolality (P<0.001). The present study also indicated that media in a range of 800 to 1,086 mOsm/kg could be assumed to be functionally iso-tonic to Pacific oysters, and osmolalities below or above this caused severe cell damage. For example, the maximum vol-ume of flagella obtained after hypotonic fixation was ap-proximately twice the volume of the flagella in isotonic fixation. Sperm cell flagellar volumes after hypertonic fixation (1,110 mOsm/kg) were 32% smaller than those in isotonic fixation, and sperm heads were 25% smaller. Al-though the damage associated with anisotonic fixation was evident in all parts of the sperm cells, the most vulnerable locations were the plasma membrane and flagellum motor apparatus. The formation of tail vesicles after hypotonic fixation was also examined. Because of water uptake, oyster sperm became swollen in hypotonic fixative, and bending or coiling of the axoneme within the tail vesicles led to the appearance of multiple axonemal structures in cross sections when observed by transmission electron microscopy. This phenomenon might be generally misinterpreted as the presence of double tails. This and other fixation artifacts can lead to the misinterpretation of damage caused by cryopreservation in ultrastructure studies of sperm of aquatic species, especially those in marine species.
The availability of tetraploid Pacific oysters provides a unique opportunity for comparative studies of sperm cryopreservation between diploids and tetraploids. In parallel to studies with sperm from diploid oysters, this study reports systematic factor optimization for sperm cryopreservation of tetraploid oysters. Specifically, this study evaluated the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), and straw size. Similar to sperm from diploids, the optimal cooling rate was 5 'C/min to -30 'C, followed by cooling at 45 'C/min to -80'C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations showed that a combination of the cryoprotectants 6% polyethylene glycol/4% propylene glycol and 6% polyethylene glycol/4% dimethyl sulfoxide yielded consistently high post-thaw motility. A long equilibration (60 min) yielded higher percent fertilization, and confirmed that extended equilibration could be beneficial when low concentrations of cryoprotectant are used. There was no significant difference in post-thaw motility between straw sizes of 0.25 and 0.5 mL. Despite low post-thaw fertilization (<10%) in general for sperm from tetraploids, optimized protocols in the present study effectively retained post-thaw motility for sperm from tetraploid oysters. This study confirmed that sperm from tetraploid Pacific oysters were more negatively affected by cryopreservation than were those of diploids. One possible explanation is that sperm from these two ploidies are different in their plasma membrane properties (e.g., structure, permeability, and elasticity), and the plasma membrane of sperm from tetraploids is more sensitive to cryopreservation effects. The fact that combinations of non-permeating and permeating cryoprotectants improved post-thaw motility in sperm from tetraploids provided presumptive evidence for this interpretation.
The water transport response during freezing of sperm cells of Morone chrysops (white bass, WB) was obtained using a shape-independent differential scanning calorimeter (DSC) technique. Sperm cell suspensions were frozen at a cooling rate of 20 C/min in two different media: (1) without cryoprotective agents (CPAs), or (2) with 5% (v/v) dimethyl sulfoxide (Me2SO). For calculations, the sperm cell was modeled as a cylinder of length 24.8 lm and diameter of 0.305 lm, while the osmotically inactive cell volume (Vb) was assumed to be 0.6 Vo, where Vo was the isotonic or the initial cell volume. By fitting a model of water transport to the experimentally determined water transport data, the best fit membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were determined, and ranged from Lpg = 0.51–1.7 · 1015 m3/Ns (0.003–0.01 lm/min-atm), and ELp = 83.6–131.3 kJ/mol (20.0–31.4 kcal/mol). The parameters obtained in this study suggest that the optimal rate of cooling for M. chrysops sperm cells is ~22 'C/min, a value that compares closely with experimentally determined optimal rates of cool-ing (~16 'C/min).
Evaluation of sperm motility is the single most widely used parameter to determine semen quality in mammals and aquatic species. While a good indicator for fresh sperm viability, post-thaw motility is not always effective at predicting fertilizing ability. Techniques using fluorescent dyes can assess functionality of mammalian sperm, but have not been widely applied in aquatic organisms. The eastern oyster Crassostrea virginica is an important mollusk in the United States, and cryopreservation protocols have been developed to preserve sperm and larvae to assist research and hatchery production. In this study, protocols were developed to assess sperm cell membrane integrity and mitochondrial function by flow cytometry and to assess viability of eggs by fluorescence microscopy. The fluorescent dyes SYBR 14 and propidium iodide (PI) (to assess membrane integrity) and rho-damine 123 (R123) (to assess mitochondrial membrane potential) were used to evaluate the quality of thawed oyster sperm previously cryopreserved with different cryoprotectant and thawing treatments. Membrane integrity results were correlated with motility of thawed sperm and mitochondrial membrane potential with fertilizing ability. Fluorescein diacetate (FDA) was used to assess cytotoxicity of cryoprotectant solutions and post-thaw damage to oyster eggs. The results indicated that membrane integrity (PD0.004) and thawing treatments (PD0.04), and mitochondrial membrane potential (PD 0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments (PD 0.0258) and with mitochondrial membrane potential (PD0.001). The dye FDA was useful in indicating structural integrity of fresh and thawed eggs. Exposure of eggs, without freezing, to dimethyl sulfoxide yielded higher percentages of stained eggs and fertilization rate than did exposure to propylene glycol (PD0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced.
Sperm cryopreservation protocols have been developed for live-bearers such as the green swordtail Xiphophorus helleri and the platyfish Xiphophorus couchianus. Despite the high post-thaw motility (∼75%) obtained in both species, the requirements of sperm storage within the female reproductive tract coupled with the process of internal fertilization place functional demands upon cryopreserved sperm samples far beyond those of oviparous species. The purpose of this study was to facilitate the artificial insemination process with cryopreserved sperm of X. helleri through evaluation of parameters related to sperm quality after thawing. Specifically, this study evaluated the effects on motility for fresh and thawed sperm samples of centrifugation (for concentration of sperm and washing for removal of cryoprotectant), ionic composition, and additions of glucose and fetal bovine serum (FBS) in extender solutions. Centrifugation at 1000 ×g for 10 min at 4 °C was found to have no adverse effects on sperm motility of fresh samples, and for cryopreserved samples, the removal of glycerol by washing yielded higher and longer post-thaw motility (e.g., 168 h vs. 48 h for the controls). Suspension of fresh sperm samples in magnesium-free Hanks' balanced salt solution (HBSS) did not affect motility; however, HBSS prepared with the absence of potassium or calcium, and the use of unsupplemented saline (NaCl alone) as extenders significantly reduced sperm motility. The presence of glucose in HBSS yielded higher and longer motility for fresh and thawed samples, but addition of glucose at greater than 2 g/L were unnecessary. Addition of 20% FBS prior to freezing was found to increase the post-thaw motility significantly compared to control treatment with 14% glycerol alone. Also addition of 20% FBS after thawing and centrifugation was found to induce the formation of sperm bundles, which may be beneficial for internal fertilization success. In conclusion, concentration of sperm and the removal of cryoprotectant (through centrifugation), and the addition of 20%FBS in the extender is recommended for future insemination trials with cryopreserved samples.
Coral species throughout the world are facing severe environmental pressures. Because of this, we began cryobiological studies on the sperm of the mushroom coral, Fungia scutaria. We determined that F. scutaria sperm had a mean length of 56 microm and head diameter of 2.5 microm, and a mean spontaneous ice nucleation temperature of -37.2 +/- 1.7 °C. When the sperm were exposed to the cryoprotectant glycerol for 5 or 20 min (at 10% v/v), no fertilized larvae were produced. However, when sperm were exposed for 20 min to propylene glycol (10% v/v), fertilizations were produced at the same rate as untreated control eggs and sperm (P > 0.05), but slightly less for dimethyl sulfoxide (10% v/v) (P < 0.05). Regardless, dimethyl sulfoxide caused less osmotic damage to the sperm membrane than did propylene glycol. Therefore, we used the dimethyl sulfoxide (10% v/v) to develop cryopreservation protocols that yielded good post-thaw morphology and motility (>95%) for coral sperm.
This study explored the optimization of techniques for sperm cryopreservation of an economically important fish species, the striped bass Morone saxatilis. The volumetric shrinkage or the water transport response during freezing of sperm cells was obtained using a differential scanning calorimeter (DSC) technique. Water transport was obtained in the presence of extracellular ice at a cooling rate of 20 8C/min in two different media: (1) without cryoprotective agents (CPAs), and (2) with 5% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of length of 22.8 mm and diameter 0.288 mm and was assumed to have an osmotically inactive cell volume (Vb) of 0.6V0, where V0 is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined water transport data, the best fit membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were determined and ranged from Lpg = 0.011–0.001 mm/min-atm, and ELp = 40.2–9.2 kcal/mol). The parameters obtained in this study suggested that the optimal rate of cooling for striped bass sperm cells in the presence and absence of DMSO range from 14 to 20 8C/min. These theoretically predicted rates of optimally freezing M. saxatilis sperm compared quite closely with independent and experimentally determined optimal rates of cooling striped bass sperm.
Experiments were performed to improve protocols for sperm cryopreservation of paddlefish (Polyodon spathula), a species for which there has been limited study. The first experiment was conducted to investigate the effects of two extenders (modified Tsvetkova’s extender: mT and modified Hanks’ balanced salt solution: mHBSS) in combination with methanol (MeOH) and dimethyl sulfoxide in two concentrations (5 and 10%) on the postthaw motility and fertilization rates of cryopreserved sperm. The highest postthaw motility (85 6 5%) was observed when sperm were frozen using mT extender with 10% MeOH as cryoprotectant. Extenders (P = 0.0018) and cryoprotectants (P = 0.0040) each had a significant effect on the postthaw motility of paddlefish sperm. The highest fertilization (80 +/- 3%) was found when eggs were fertilized with sperm frozen with mT extender in combination with 10% MeOH. However, there was no significant difference among fertilization rates when MeOH was used as a cryoprotectant in either concentration or in combination with either mT or mHBSS extenders. In the second experiment, 4000 eggs were fertilized with the pooled contents of five straws of thawed sperm (total volume of 1.25 mL) using mT extender in combination with 5% MeOH, and hatch rates as high as 79 +/- 5% were observed. A third experiment was also conducted to clarify the role of MeOH concentration; however, no significant difference was found among fertilization and hatch rates when either 5 or 10% MeOH was used as a cryoprotectant. These results suggest that MeOH is a safe and reliable cryoprotectant for freezing of paddlefish sperm and obtaining viable postthaw sperm for consistent fertilization and hatch rates. Further, this experimental protocol is relatively simple and applicable for commercial hatchery production of paddlefish.
A lack of standardization in sperm cryopreservation of aquatic organisms is one of the main reasons for inconsistency observed among various studies. In particular, there have been few attempts to standardize sperm concentration during procedural optimization. This study was intended to call attention to sperm concentration standardization through research of sperm agglutination in Pacific oysters Crassostrea gigas. Sperm agglutination after thawing is a relatively frequent phenomenon observed for various aquatic species, especially when sub-optimal cryopreservation protocols are used; however, no systematic attempts have been made to explain this phenomenon. The present study evaluated various factors affecting sperm agglutination of thawed samples from diploid and tetraploid PaciWc oysters, and is the first detailed report addressing the sperm agglutination phenomenon of thawed samples from any aquatic organism. Agglutination of oyster sperm was classified into six levels with a scale ranging from 0 (homogenous suspension) to 5 (well-developed “noodles”). It was found that agglutination in thawed samples was mainly due to the lack of sufficient cryoprotectant for a specific sperm concentration. Interestingly, high levels of agglutination did not necessarily lead to low fertilization. On the contrary, some sperm cells appeared to gain protection from the formation of peripheral agglutination within 0.5-ml French straws. The exact mechanism of sperm agglutination remains unclear. However, morphological examination of cross sections of the noodles (agglutination level 5) indicated at least two forms of agglutination (formed with and without cryoprotectant) which could be used as a tool to understand the cryopreservation process within the micro-environment of the straw. Furthermore, the fact that the level of sperm agglutination was directly determined by sperm concentration, in addition to the type of cryoprotectant, cryoprotectant concentration, and cooling and thawing methods emphasized the importance of procedural standardization and systematic optimization and integration of protocols involving multiple factors.
Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation proto-cols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.
This study documents early out-of-season induced spawning of channel catfish Ictalurus punctatus. During the early spring (February-April) of 1999, 2000 and 2001, ponds containing (1) male and female channel catfish (mixed-sex ponds) or (2) male channel and blue catfish I. furcatus only, or female channel catfish only (single-sex ponds) were heated to 24^ 30 1C to encourage gonadal maturation and spawning. Unheated ponds were stocked with males and females and were monitored during the duration of heating. When natural spawning occurred in the heated ponds, the fish were captured by seining and unspawned females were injected with 100 mgkg^-1 of synthetic leutenizing hormone-releasing hormone. Injected females were either paired with males or held in communal all-female groups, and monitored for ovulation. Eggs were collected and fertilized with sperm of channel catfish or blue catfish. Females paired with males were induced to spawn 44 days (mixed-sex ponds) and 50 days (single-sex ponds) be-fore natural spawning occurred in unheated ponds. Spawning latency (the time between injection and ovulation) and the percentage of neurulated embryos from eggs fertilized using channel cat¢sh sperm was not different between spawning before the natural season (P 5 0.68) and during the natural season in¢sh from mixed-sex ponds (P 5 0.57). Females held in all-female groups produced eggs 34 days before the onset of spawning in unheated ponds. Spawning latency was not different between spawns before and during the natural season (P 5 0.16), and the percentages of neurulated embryos from eggs fertilized with channel cat¢sh sperm (P 5 0.76) or blue catfish sperm (P 5 0.77) before or during the natural season were not different. This study demonstrates the feasibility of conditioning of channel catfish females for early out-of-season induced spawning in the laboratory.
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.
Sperm cryopreservation offers potential for long-term storage of genetic resources. However, the current protocols for zebrafish Danio rerio are cumbersome and poorly reproducible. Our objective was to facilitate adoption of cryopreservation by streamlining methods from sperm collection through thawing and use. First, sperm activation was evaluated, and motility was completely inhibited when osmolality of the extender was >=295–300 mOsmol/kg. To evaluate cryoprotectant toxicity, sperm were incubated with dimethyl sulfoxide (DMSO), N,N-dimethyl acetamide (DMA), methanol, or glycerol at 5, 10, and 15% concentrations. Based on motility, DMSO, DMA, and methanol (10%) were less toxic; therefore, sperm were cryopreserved using these cryoprotectants at cooling rates of 10 and 20 8C/min. The highest motility (mean +/- S.D.) (35 +/- 23%; P = 0.0001) and fertility (13 +/- 8%; P = 0.001) in thawed sperm were obtained with the combination of 8% methanol and a cooling rate of 10 'C/min. Further evaluations of 8% methanol and 10 'C/min were performed with males from populations with high (2.05 +/- 0.24) and low (1.18 +/- 0.12) body condition (P = 0.0001). Motility of thawed sperm from the two populations was 38 16% (range, 10 to 60%) and 78 +/- 10% (50 to 90%) (P = 0.0001), and fertilization was 6 +/- 6% (0 to 18%) and 33 +/- 20% (5 to 81%) (P = 0.0001). These values were positively related with body condition factor. Overall, this study simplified and standardized sperm cryopreservation, and established a protocol using French straws as a freezing container and an extender without powdered milk. This protocol can be readily adapted for high-throughput application using automated equipment, and motility and fertility comparable to previous reports were obtained. Male variability and sperm quality remain important considerations for future work, especially in mutant and inbred lines.
The consequences of UVB and UVA irradiation on hatch rate, mortality, and malformation were studied in embryonic zebrafish (Danio rerio). The use of zebrafish embryos has expanded from traditional developmental models to diverse studies, including many techniques utilizing light exposure. To characterize useful indicators of photodamage, the responses and threshold limits of UV radiation as a function of embryonic stage and fish source were evaluated. Significant differences in UVB susceptibility were observed in embryos at 3, 6–7, 12, and 24 h post-fertilization (hpf), with the 1000-cell stage (3 hpf) having greatest tolerance to UVB. Embryos derived from zebra-fish raised in outdoor ponds were more tolerant to UVB than were embryos from laboratory-raised fish. Combinations of UVB and UVA exposure were used to confirm the presence of a competent photorepair system in zebrafish that could return otherwise malformed embryos to a normal phenotype. Overall, embryonic zebrafish had large tolerances (LD50 of 850 J/cm2) to UVA, confirming their suit-ability for photoactivation and photorepair studies.
Although sperm cryopreservation has been practiced in aquatic species for more than 50 years, viable markets for frozen sperm do not currently exist for commercial aquaculture. The present study suggests that the use of commercial cryopreservation facilities used for dairy bulls could be a cost-effective approach to initiate commercialization of frozen sperm in aquaculture, and the oyster industry could become one of the early adopters. To prove the technical feasibility of the use of a commercial freezing facility, this study adopted dairy freezing methods and emphasized cryoprotectant optimization for sperm from diploid Pacific oysters Crassostrea gigas with specific cooling methods employed for use with bull sperm. Specifically, the present study evaluated dimethyl sulfoxide (DMSO) at 5, 8, and 10%, ethylene glycol (E-glycol) at 2, 5, 8, and 10%, and methanol at 2, 4, 6, and 8%. Each cryoprotectant with its optimal concentration was chosen for subsequent selection of an optimal cryoprotectant. Previous results showed propylene glycol (P-glycol) at 5% yielded higher percent fertilization than did PG at 10 or 15%. Therefore, 5% of these cryoprotectants were compared and the highest percent fertilization was obtained with methanol (49 ± 29%), followed by E-glycol (42± 15%), DMSO (31 ± 18%), and P-glycol (22 ± 12%). Extensive evaluation for single and combined cryoprotectants and their concentrations were studied in our previous trials on a research scale (reported elsewhere), and 6% methanol and the combination of 4% methanol and 2% polyethylene glycol (PEG; FW 200) were shown to consistently yield the highest percent fertilization. Our last commercial-scale experiment compared 6% methanol with the combination of 4% methanol and 2% PEG (MET/PEG) with 20 oysters. There was no significant difference for percent fertilization between 6% methanol (39 ± 29%) and 4% MET/2% PEG (42 ± 26%). These findings demonstrate the technical feasibility of adopting dairy freezing protocols in commercial application for oyster sperm, and also provide a template for future commercialization of sperm cryopreservation for other aquatic species.
We sought to develop sperm cryopreservation methods for the pallid sturgeon Scaphirhynchus albus,a federally listed endangered species. Males were injected with synthetic luteinizing hormone releasing hormone at 50 lg/kg of body weight. After 24 h, sperm were collected, diluted at a ratio of 1:4 (sperm : extender) with Hanks’ balanced salt solution (HBSS; diluted to 100 milliosmoles/kg), and kept refrigerated until use. Methanol was used as a cryoprotectant at concentrations of 5, 10, and 15% (volume per volume) and was mixed 1:1 with HBSS before the experiment to reduce effects of initial mixing. Sperm were mixed with the cryoprotectant, loaded into 0.5-mL straws, packed into goblets (5 straws/goblet), and placed in the lower position on aluminum canes. Motility was estimated before freezing to determine the effects of cryoprotectant toxicity; there was no significant difference in motility at the concentrations tested (P = 0.4828). After a 2-min equilibration period, the canes were lowered into a nitrogen vapor shipping dewar. The cooling rate of -22'C/min was recorded by thermocouples inserted into 0.5-mL straws filled with extender and cryoprotectant. After 1 year of storage in liquid nitrogen, straws were thawed in a 40'C water bath for 9 s and motility was estimated. Postthaw motility did not differ among the cryoprotectants tested (P = 0.4880). Each sample was used to fertilize approximately 150 eggs, which were incubated at 218C. Sperm that was cryopreserved with 5% or 10% methanol produced eggs with significantly higher hatch rates did sperm cryopreserved with 15% methanol (P = 0.0001). The development of techniques for cryopreserved sperm of pallid sturgeon allows for the creation of germplasm repositories that will aid in the recovery of this endangered species.
Red snapper Lutjanus campechanus and gray snapper L. griseus support valuable sport and commercial fisheries. Because of their high market value and limited commercial harvests, these species are prime candidates for aquaculture and stock enhancement. Development of culture techniques for snapper species has been attempted over the past 30 years, but use of gonadotropic hormones with mature, wild-caught red snapper remains the most reliable method for inducing ovulation and producing eggs that can be fertilized. In this study, procedures for sperm collection, handling, and refrigerated storage were developed to improve strip-spawning techniques for these snapper species. Use of refrigerated sperm allows efforts to be focused on maintaining female broodstock, monitoring of ovarian development, and increasing efficiency during the strip-spawning process. Sperm were collected from male red snapper (n = 199) and gray snapper (n = 83) captured in the recreational fishery during the summers of 2000 and 2001. Sperm were diluted 1:4 with calcium-free Hanks’ balanced salt solution (HBSS), placed in 4-L plastic bags, and transported to the laboratory on ice. Osmotic pressure (mean +/- SE) of seminal plasma was 428 +/- 15 milliosmoles (mOsm) per kilogram for red snapper (n = 19) and 411 +/- 5 mOsm/kg for gray snapper (n = 13). Blood plasma osmolality was 440 +/- 7 mOsm/kg for red snapper and 421 +/- 7 mOsm/kg for gray snapper. Activation studies of red and gray snapper sperm indicated that sperm motility was suppressed by decreasing the osmotic pressure of artificial seawater to a level less than 400 mOsm/kg. Refrigerated storage experiments demonstrated that sperm samples suspended in 200-mOsm/kg HBSS retained motility for 10 d when refrigerated at 48C. These results show that red snapper and gray snapper sperm can be stored for short-term repeated use in a hatchery.
Swordtails and platyfish of the genus Xiphophorus are viviparous teleost of the family Poecilliidae.
They are valuable models for biomedical research, especially for cancer genetics. This animal
model was one of the first to prove that certain cancers are inherited diseases. In addition, they are also
valued as ornamental fish for the aquarium trade because of vibrant body coloration and a long sword-like
tail (in males). Species of this genus usually attain sexual maturity at 10-12 weeks of age [l]. The sperm
of Xiphophorus fishes are different in structure (e.g., head shape) and physiology (e.g., energy metabolism)
from the sperm of oviparous fishes. Sperm of internally fertilizing species possess atypical features such
as well-developed mitochondrial sheaths in the midpiece of spermatozoa and glycolytic activity comparable
to that of mammalian sperm, which may be adaptations for movement or long-tenn survival in the female
reproductive tract.
The increased research and commercial value, and the continuous decline of diversity in the wild
of these fish, expand the need to preserve their genetic resources. Despite study of sperm cryopreservation
in some 200 species of freshwater and marine fishes, sperm cryopreservation has just begun in live-bearers
including Xiphophorus [2]. Cryopreservation protocols have been developed to address their small body
size and limited sperm volume ( e.g., 5-10 µl per fish), and post-thaw motility as high as> 70% has been
observed withX helleri [3] and X. couchianus [4]. Recently, live young were produced with cryopreserved
sperm fromX helleri (our unpublished data).
The Pacific oyster, Crassostrea gigas (Thunberg, 1793), also referred to as the Japanese oyster or giant
oyster, is one of the most important species of bivalves cultured worldwide. The basic life history of
the Pacific oyster is similar to that of the eastern oyster, C. virginica [ 1] with both functioning as
protandrous hermaphrodites. The young are functionally male during their first spawning, while adults
function as separate male or female animals in any given reproduction season [2]. Sexual maturity is
reached during the first year, and spawning generally occurs in the summer months ( June to August in
the west coast of United States). Oysters are highly prolific, and males generally produce 5 x 10^10 sperm
per gram of gonad wet weight [3].
The first report of sperm cryopreservation in oysters was published [ 4] with C. virginica.
Since then some 27 reports have been published to address oyster sperm cryopreservation, of which ~80%
have addressed sperm from the Pacific oyster. Despite that, procedural standardization is lacking in the
cryopreservation of oyster sperm [5]. Inconsistency of various components of cryopreservation
technology have been observed among and within studies, such as initial sperm quality, gamete collection
methods, extender formulation, cryoprotectant choice, cooling rate and method, thawing rate and method,
insemination protocols, and evaluation of postthaw sperm quality [5]. The procedures outlined below
are mainly derived from our 4 years of research on this species.
Crassostrea virginica (formerly the "American oyster") was designated in 1985 as the "eastern oyster" by the Committee on Scientific and Vernacular Names of Mollusks of the Council of Systematic Malacologists [l]. The eastern oyster is a lamellibranch with pronounced bilateral asymmetry [2] and usually spawns as a male in the first year, a condition called protandry. Fecundity has been estimated to be between 500,000 and 66 million eggs per female depending upon body size [3,4]. Within 8 to 12 h (depending on temperature) fertilized eggs develop into freeswimming larvae or trochophores (50 to 60 µm in width). After-24 h, the trochophore larvae develop into veliger or D-stage larvae (70 to 12 5 µm). After metamorphosis, the settled larvae develop into adults within 1 to 2 years. Adult oysters mature and spawn in the summer months (April to September in the Gulf of Mexico) for the next cycle [5,6]. The eastern oyster is the most important bivalve species in the United States [7]. However, along the Atlantic and Gulf coasts, oyster production has declined over the past century due to reasons including a lack of consistent seed supply, excessive harvest, loss of suitable habitat, disease, and natural predation [8]. The use of cryopreserved gametes and larvae can improve hatchery production of seedstock to increase production for the oyster industry. Cryopreservation of oyster sperm and larvae has been tested at the laboratory level, but given the benefit that this technique offers, cryopreservation of oyster sperm and larvae should be developed for commercial application at the hatchery level. The procedures outlined below are suitable for application at the hatchery scale, and could be scaled up for commercial application.
Temperature and time are critical in determining when channel catfish Ictalurus punctatus will be physiologically ready to spawn (conditioned). Natural and hormone-induced spawning can occur when the fish are held at, above, or below specific temperatures for a largely undetermined minimum period of time. This period of conditioning can also be described as the heating requirement for spawning. We used degree-days as a standard unit to describe and quantify the heating requirement for channel catfish spawning, and we developed specific terminology and standard parameters to support the use of the degree-day concept for channel catfish spawning. The heating requirements for spawning were quantified by (1) retrospectively calculating degree-day values using previously collected data (2000–2003) from heated and ambient-temperature ponds before and during the natural spawning season and (2) calculating degree-days using data from fish conditioned for the 2004 natural spawning season at different thermal regimes in heated ponds at three threshold temperatures. These results were used to calculate spawning probabilities to enable future spawning predictions using degree-days. The values calculated using the 218C threshold were more consistent than those calculated using the two other thresholds: 188C and 248C. The heating requirements for spawning in heated ponds before the natural spawning season were not different from the requirements for spawning in ambient-temperature ponds during the natural spawning season. Degree-day guidelines for channel catfish spawning were proposed based on the results of the degree-day calculations and spawning probabilities using a commercial definition that corresponded with the 218C threshold. According to those guidelines, channel catfish spawning can begin in 0.04-ha ponds in southern Louisiana after the accumulation of 57–172 degree-days, with a median value of 99 degree-days.
Populations of sturgeon across the globe are threatened due to unregulated harvest and habitat loss, and the status varies among species across North America. Ready access to viable and functional sperm would contribute to recovery pro-grammes for these species. In this study, we examined the motility, viability (cell membrane integrity) of cryopreserved sperm from three North American acipenseriform species and fertilizing capacity. Milt samples were collected from captive shortnose sturgeon (Acipenser brevirostrum), wild paddlefish (Polyodon spathula) and pallid sturgeon (Scaphirhynchus albus) and cryopreserved using combinations of Modified Tsvetk-ova's (MT) extender, Original Tsvetkovas extender, and modified Hanks' balanced salt solution, along with the cryoprotectants methanol (MeOH) or dimethyl sulfoxide (DMSO). A dual-staining technique using the fluorescent stains SYBR-14 and propidium iodide was employed with flow cytometry to determine the percentages of spermatozoa that were viable by virtue of having intact membranes. The percentage of viable spermatozoa ranged from 5% to 12%in shortnose sturgeon, 30–59% in paddlefish, and 44–58% in pallid sturgeon. In the first experiment with shortnose sturgeon sperm, methanol allowed for higher values for dependent variables than did DMSO, and sperm viability generally correlated with post-thaw motility. However, fertilization rate, neurulation, or hatching rates were independent from these factors. In the second experiment with shortnose sturgeon, 5%MeOH combined with MT yielded higher values for all parameters tested than the other combinations: viability was correlated with motility, fertilization rate, and hatching rate. Overall, viability and post-thaw motility was not affected by the use of hyperosmotic extenders (OT) or cryoprotectants (DMSO), but their use decreased fertilization percentages. For paddlefish sperm (experiment 3), MT combined with 10%MeOH was clearly a good choice for cryopreservation; viability and motility results were correlated, but independent of fertilization. For pallid sturgeon sperm (experiment 4), MT with 5–10% MeOH showed significantly higher sperm quality and fertilization parameters. Membrane integrity can be used as a predictor of fertilization by cryopreserved sperm, however additional sperm quality parameters, supplementary to motility and membrane integrity, would be useful in the refining and optimizing cryopreservation protocols with acipenseriform sperm.
DNA photorepair has been widely studied in simple aquatic organisms that live in the marine environment, but is less understood in more complex species that live in freshwater. In the present study, we evaluated UVA-induced DNA photo recovery in embryonic stages of zebrafish, Danio rerio, a freshwater model species. Evaluation of UVB exposure and UVA photo recovery of zebrafish embryos revealed different UVB tolerances and capacities for UVA photo recovery at different stages of development. Effective UVA photo recovery was observed at 3 h post-fertilization (hpf), 6–7 hpf, and 12 hpf, but not in the early cleavage stage (2–32 cells). UVA photo recovery was most effective during the gastrula stage (6–7 hpf) of development, and less effective at earlier stages (e.g., 3 hpf) or later stages (e.g., 12 hpf). Embryos at the cleavage stage of development were found to be tolerant to extreme levels of UVB exposure, and possible mechanisms were discussed. For embryos at 6–7 hpf, examination of time window (or delay of UVA exposure) that would still permit recovery from UVB exposure suggested a short time period of 2 h. The transgenic fli-1 zebrafish with fluorescent vascular structure was used to show that embryos with normal morphological appearance could exhibit a disrupted vascular patterning, suggesting that this endpoint could provide a sensitive tool for detection of UV damage.
Initial success in sperm cryopreservation occurred 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 cryopreserved sperm of aquatic species remains a research activity with little commercial application despite work in more than 90 species and more than 200 published reports. Most research work has focused on large-bodied culture and sport fishes, such as salmon, trout, carp, and catfish, and mollusks such as commercially important oyster and abalone species. However, only a few studies have addressed sperm cryopreservation in small fishes such as zebrafish, or in endangered species. Overall, this work has yielded techniques that are being applied with varied 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, a 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 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, and appropriate biosecurity safeguards.
In this work Intracytoplamic Sperm Injection (ICSI) was evaluated in three genera of finfish. The different characteristics that must be considered when performing ICSI include: spawning period and conditions, gamete size and activation characteristics, adhesive properties of eggs, transparency of the chorion, size and localization of the micropyle, and the value of the species studied.
In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (Vo) of 12.1 ± 0.2 lm^3 (SEM), a water permeability (Lp) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) lm/min/atm, and a cryoprotectant permeability (Ps) of 0.10 ± 0.01 (SEM) x 10^-3 cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0 'C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.
Fishes of the genus Xiphophorus are well-studied biomedical research models, and some species, such as X. couchianus, are currently listed as endangered in the wild. Sperm cryopreservation in these live-bearing fishes has begun recently. Thus far, live young have been produced with cryopreserved sperm only in one species (Xiphophorus helleri). In this study, the goal was to develop a practical protocol for sperm cryopreservation of Xiphophorus couchianus, and to produce live young with cryopreserved sperm. Sperm were collected by crushing of testis in Hanks' balanced salt solution at an osmolality of 500 mOsmol/kg (HBSS500), and were cryopreserved with 14% glycerol (v/v) as cryoprotectant at a cooling rate of 20 °C/min from 5 to −80 °C in 250-μL French straws. For artificial insemination, samples were thawed at 40 °C for 5 s in a water bath, washed once using fresh HBSS500 by centrifuging at 1000 g for 5 min at 4 °C, concentrated into ~5 μL, and injected into virgin females of Xiphophorus maculatus. The inseminated females were monitored for 90 days for subsequent discharge of live young. Results from 2006 and 2007 showed considerable maleto- male variation in post-thaw motility (from 1 to 70%). Offspring were produced by cryopreserved sperm in two tanks (of three) at 36 and 66 days after insemination in 2007. Paternity was confirmed via phenotypes (body color) and genotypes (microsatellite genetic marker) of the hybrid offspring. Overall, a practical protocol for sperm cryopreservation and artificial insemination is provided to preserve X. couchianus, which is an important biomedical research model, and also currently listed as an endangered species in the International Union for Conservation of Nature (IUCN) red list.
Aquarium fishes are becoming increasingly important because of their value in biomedical research and the ornamental fish trade, and because many have become threatened or endangered in the wild. This review summarizes the current status of sperm cryopreservation in three fishes widely used in biomedical research: zebrafish, medaka, and live-bearing fishes of the genus Xiphophorus, and will focus on the needs and opportunities for future research and application of cryopreservation in aquarium fish. First, we summarize the basic biological characteristics regarding natural habitat, testis structure, spermatogenesis, sperm morphology, and sperm physiology. Second, we compare protocol development of sperm cryopreservation. Third, we emphasize the importance of artificial fertilization in sperm cryopreservation to evaluate the viability of thawed sperm.We conclude with a look to future research directions for sperm cryopreservation and the application of this technique in aquarium species.
The medaka, Oryzias latipes, is a well-recognized fish model for biomedical research. An understanding of gamete characteristics is necessary for experimental manipulations such as artificial fertilization and sperm cryopreservation. The goal of this study was to investigate sperm characteristics of motility initiation, duration, and retention in medaka. First, motility was initiated by osmolality values ranging from 25 to 686 mOsm/kg, which included deionized water and hypotonic, isotonic, and hypertonic Hanks’ balanced salt solution. The percentage of motile sperm was >80% when osmolality was <315 mOsm/kg and decreased as osmolality increased. This is different from most fish with external fertilization in which sperm motility can be initiated by hypotonic (for freshwater fish) or hypertonic (for marine fish) solutions or by altering the concentration of specific ions such as potassium (e.g., in salmonids). Second, upon activation, the sperm remained continuously motile, with reserve capacity, for as long as 1 wk during storage at 4'C. This was also different from other externally fertilizing fish, in which motility is typically maintained for seconds to several minutes. Third, after changing the osmolality to 46 to 68 mOsm/kg by adding deionized water, the motility of sperm held at 274 to 500 mOsm/kg was higher than the original motility (P = 0.035) after 24, 48, and 72 h of storage at 4'C. Fourth, the addition of glucose had no effect on maintaining sperm motility during refrigerated storage. To our knowledge, this combination of sperm motility characteristics is reported for the first time in fish and may be unique to medaka or may represent an undescribed modality of sperm behavior within euryhaline fish.
The goal of this study was to establish an efficient method for determination of sperm concentration requiring only 1–2 mL of sample by use of microspectrophotometry. The objectives were (1) determination of wavelengths with absorbance profiles appropriate for analysis of sperm suspensions from zebrafish Danio rerio, green swordtail Xiphophorus helleri, and medaka Oryzias latipes collected by crushing of dissected testis or by stripping of live males; (2) generation of standard curves and equations between sperm sample absorbance and sperm concentration estimated by hemocytometer counts; (3) accuracy verification of equations for estimating concentration by microspectrophotometry; and (4) analysis of the precision in generating equations and estimation of sperm concentration. Within the visible wavelengths (380–750 nm) there was no single maximal absorbance peak. For zebrafish, a linear correlation was established with an effective absorbance range of 0.034–0.936 for crushed samples, and 0.028–0.961 for stripped samples at 400 nm. For Xiphophorus, the effective absorbance range was 0.014–1.154 for crushed samples, and the effective range was 0.038–1.082 for stripped samples. For medaka, the effective range was 0.041–0.896 for crushed samples. The accuracy of these equations was verified by comparison of sample concentrations counted with hemocytometer and calculated with equations, and no significant differences ( p ¼ 0.447) were observed. Measurement of serially diluted aliquots from pooled samples verified the precision of techniques used. Overall, this confirmed that microspectrophotometric estimation of sperm concentration is accurate, efficient, and sample-saving for use with small-bodied fishes.
Medaka Oryzias latipesis a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modication. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: (1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me 2SO),N,N-dimethylacetamide (DMA), N,Ndimethyl formamide (DMF), and glycerol with concentrations of 5%, 10%, and 15% for 60 min of incubation at 4 C; (2) evaluate cooling rates from 5 to 25 C/min for freezing and their interaction with cryoprotectants, and (3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5% and 10%) did not change the sperm motility after 30 min; Me 2SO, DMA, and DMF (10% and 15%) and glycerol (5%, 10% and 15%) signi“cantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 to 25 C/min) and were compared to Me 2SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction ( P6 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was 6 10% which was significantly lower than that of methanol and ME. With Me 2SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants ( P6 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 C/min and 10% ME with a rate of 15 C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.
Flow cytometry (FCM) and computer-assisted sperm motion analysis (CASA) methods were developed and validated for use with endangered razorback suckers Xyrauchen texanus collected (n=64) during the 2006 spawning season. Sperm motility could be activated within osmolality ranges noted during milt collections (here 167–343 mOsm/kg). We hypothesized that sperm quality of milt collected into isoosmotic (302 mOsm/kg) or hyperosmotic (500 mOsm/kg) Hanks’ balanced salt solution would not differ. Pre-freeze viabilities were similar between osmolalities (79% +/- 6(S.E.M.) and 76% +/- 7); however, post-thaw values were greater in hyperosmotic buffer (27% +/- 3 and 12% +/- 2; P=0.0065), as was mitochondrial membrane potential (33% +/- 4 and 13%+/- 2; P=0.0048). Visual estimates of pre-freeze motility correlated with total (r=0.7589; range 23–82%) and progressive motility (r=0.7449) by CASA and were associated with greater viability (r=0.5985; P=0.0001). Count (FCM) was negatively correlated with post-thaw viability (r=-0.83; P=0.0116) and mitochondrial function (r=-0.91; P=0.0016). By FCM-based assessments of DNA integrity, whereby increased fluorochrome binding indicated more fragmentation, higher levels were negatively correlated with count (r=-0.77; P=0.0001) and pre-freeze viabilities (r=-0.66; P=0.0004). Fragmentation was higher in isotonic buffer (P=0.0234). To increase reproductive capacity of natural populations, the strategy and protocols developed can serve as a template for use with other imperiled fish species, biomonitoring, and genome banking.
The present study focused on development of a sperm cryopreservation protocol for the critically endangered olive barb Puntius sarana (Hamilton, 1822) collected from two stocks within Bangladesh and reared in the Fisheries Field Laboratory, Bangladesh Agricultural University (BAU). The sperm were collected in Alsever’s solution prepared at 296 mOsmol kg1. Sperm were activated with distilled water (24 mOsmol kg1) to characterize motility. Maximum motility (90%) was observed within 15 s after activation, and sperm remained motile for 35 s. Sperm activation was evaluated in different osmolalities and motility was completely inhibited when osmolality of the extender was P287 mOsmol kg1. To evaluate cryoprotectant toxicity, sperm were equilibrated with 5%, 10% and 15% each of dimethyl sulfoxide (DMSO) and methanol. Sperm motility was noticeably reduced within 10 min, when sperm were equilibrated with 15% DMSO, indicating acute toxicity to spermatozoa and therefore this concentration was excluded in further trials. Sperm were cryopreserved using DMSO at concentrations of 5% and 10% and methanol at 5%, 10% and 15%. The one-step freezing protocol (from 5 C to 80 C at 10C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL CL-3300; Australia) and 0.25-ml straws containing spermatozoa were stored in liquid nitrogen for 7–15 days at 196 C. The highest motility in thawed sperm 61 ± 8% (mean ± SD) was obtained with 10% DMSO. The fertilization and hatching rates were 70% and 37% for cryopreserved sperm, and 72% and 62% for fresh sperm. The protocol reported here can be useful for hatchery-scale production of olive barb. The use of cryopreserved sperm can facilitate hatchery operations, and can provide for long-term conservation of genetic resources to contribute in the recovery of critically endangered fish such as the olive barb.
Hybrid catfish created by crossing of female channel catfish (Ictalurus punctatus) and male blue catfish (Ictalurus furcatus) are being used increasingly in foodfish aquaculture because of their fast growth and efficient food conversion. However, the availability of blue catfish males is limited, and their peak spawning is at a different time than that of the channel catfish. As such, cryopreservation of sperm of blue catfish could improve production of hybrid catfish, and has been studied in the laboratory and tested for feasibility in a commercial dairy bull cryopreservation facility. However, an approach for commercially relevant production of cryopreserved blue catfish sperm is still needed. The goal of this study was to develop practical approaches for commercial-scale sperm cryopreservation of blue catfish by use of an automated high-throughput system (MAPI, CryoBioSystem Co.). The objectives were to: (1) refine cooling rate and cryoprotectant concentration, and evaluate their interactions; (2) evaluate the effect of sperm concentration on cryopreservation; (3) refine cryoprotectant concentration based on the highest effective sperm concentration; (4) compare the effect of thawing samples at 20 or 40'C; (5) evaluate the fertility of thawed sperm at a research scale by fertilizing with channel catfish eggs; (6) test the post-thaw motility and fertility of sperm from individual males in a commercial setting, and (7) test for correlation of cryopreservation results with biological indices used for male evaluation. The optimal cooling rate was 5 C/min (Micro Digitcool, IMV) for high-throughput cryopreservation using CBS high-biosecurity 0.5-ml straws with 10% methanol, and a concentration of 1 x 10^9 sperm/ml. There was no difference in post-thaw motility when samples were thawed at 20'C for 40 s or 40'C for 20 s. After fertilization, the percentage of neurulation (Stage V embryos) was 80 ± 21%, and percentage of embryonic mobility (Stage VI embryo) was 51 ± 22%. There was a significant difference among the neurulation values produced by thawed blue catfish sperm, fresh blue catfish sperm (P = 0.010) and channel catfish sperm (P = 0.023), but not for Stage VI embryos (P>=0.585). Cryopreserved sperm from ten males did not show significant variation in post-thaw motility or fertility at the neurulation stage. This study demonstrates that the protocol established for high-throughput cryopreservation of blue catfish sperm can provide commercially relevant quantities and quality of sperm with stable fertility for hybrid catfish production and provides a model for establishment of commercial-scale approaches for other aquatic species.
The use of fluorescent staining in conjunction with flow cytometry for the assessment of sperm quality in
aquatic species has increased over the past decade. However, in contrast to the common use of flow
cytometric assessment of sperm quality in mammalian species (i.e., hundreds of published studies), the
use of this technique in aquatic species is relatively unexploited but becoming increasingly important.
The increase in usage has been largely driven by the adaptation and adoption of sperm quality assays
developed for use in mammalian species, which are vast in number compared to the range currently applied
in aquatic species (Martinez-Pastor et al. 2010). Most assays developed in mammalian species are designed
to target specific sperm structures or processes that are often sufficiently conserved across species to
allow application in aquatic species. In addition to the assays adapted from mammalian species, more
specialized sperm quality assays have been optimized in aquatic species for biomonitoring applications
(e.g., Jenkins et al. 2010) and these have great potential for use in sperm quality assessments for
cryopreservation.
Among the sperm quality assays that are currently used in aquatic species, assays for sperm membrane
integrity and mitochondrial membrane potential have had the greatest application. One of the most commonly
used in aquatic species is the SYBR 14/ propidium iodide (PI) assay for plasma membrane integrity, often
referred to as a “sperm viability” assay. Several studies have used this assay to assess sperm quality
in fresh and post-thaw samples to evaluate the success of cryopreservation protocols (e.g., Cabrita et
al. 2005, Paniagua-Chávez et al. 2006). This assay has also been used in environmental toxicity studies
to determine the effect of exposing sperm to antibiotics (Segovia et al. 2000) and herbicides (Favret and
Lynn 2010) in a range of vertebrate and invertebrate aquatic species. Another commonly tested parameter
in sperm from aquatic species is mitochondrial function, which is assessed using mitochondrion-specific
fluorescent dyes that accumulate in proportion to membrane potential. The most commonly used mitochondrial
dye in aquatic species is rhodamine 123, which is often combined with PI to enable concurrent assessment
of membrane integrity (e.g., Ogier de Baulny et al. 1997, Liu et al. 2007), but a few studies have used
other mitochondrial stains such as MitoTracker Red (Favret and Lynn 2010) and JC-1 (Guthrie et al. 2008).
In addition to the often kit-based assays adapted from mammalian species, assays that are currently
used in aquatic species for biomonitoring applications also have potential utility in sperm quality
assessments for cryopreservation, including assessment of DNA integrity (Jenkins et al. 2010) and ploidy
analysis (Psenicka et al. 2009). These assays use fluorescent dyes that label DNA, such as PI or
4',6-diamidino-2-phenylindole (DAPI), and are typically used to assess the effect of environmental toxins
on spermatogenesis and subsequent sperm quality. Although they require a greater level of flow cytometric
expertise than the kit-based assays and require research for validation studies, they offer additional
means of assessing and predicting the effect of cryopreservation on sperm survival and fertilizing ability.
Computer-assisted sperm analysis (CASA) (also referred to as computer-assisted semen analysis) uses computer
software to collect, detect, identify, and quantify attributes of motility in a sperm sample. It was first
designed for use in humans and livestock, and is considered to be an objective, accurate approach for
sperm motility assessment in mammals because it relies on actual counts and measurements rather than
subjective observation and estimation. Basically, these systems comprise three components: 1) an optical
system; 2) a method for image capture, and 3) data analysis and reporting. Currently, several manufacturers
provide complete CASA systems or software (listed in Table 1). Based on the descriptions from the
associated user manuals, the basic functions of these systems are similar and are typically based on
quality standards developed for human semen put forth by the World Health Organization (World Health
Organization 2010).
To produce accurate and reliable results by use of CASA, a series of parameters and thresholds
in the system need to be properly established to ensure that sperm cells can be recorded and sorted into
appropriate categories such as cell size, contrast, and identification of movement. These settings are
essential for the application of CASA, and are based on characteristics such as size, shape, and swimming
trajectory of sperm from each species. For most CASA systems, these settings can be validated by playing
back of videos in sequence and inspecting the frames in real time to confirm if the cells were categorized
correctly.
Maintenance of rare or valuable genetic material is vital for preservation of biomedical research
resources derived from model organisms such as zebrafish Danio rerio and medaka Oryzias latipes.
However, the aquarium fish research community is becoming overwhelmed by the maintenance of thousands
of research lines as live populations. Cryopreservation is a proven method for large-scale preservation
and maintenance of important genetic material. It extends the reproductive potential of males, reduces
the need to maintain live fish, and can prevent catastrophic loss of irreplaceable research lines.
Cryopreservation is the most cost-effective alternative for maintaining genetic resources of aquatic
organisms, because it can reduce costs for fish and facility maintenance, personnel, and space, and
accelerate the development of new research lines.
A cryopreservation program requires post-thaw evaluation of sperm quality on a per sample basis.
This challenge is magnified when working with a high-throughput system, when non-wild-type strains are
being cryopreserved, and when standardization is required among laboratories. Currently the most commonly
used method for estimating quality of fish sperm is evaluation of motility. This has been performed
qualitatively by microscopic observation, but now increasingly is performed by use of computer-assisted
semen analysis (CASA) systems originally designed for use with human and livestock sperm. Unfortunately,
zebrafish sperm can lose peak motility rapidly after activation (within 10 sec) and existing CASA
systems cannot begin to capture data rapidly enough to reliably monitor the peak motility phase. This
problem is exacerbated by use of volumetric viewing chambers that are slow to fill and are subject to
swirling currents that can interfere with the CASA analysis. These challenges have been addressed by
performing CASA analysis with open glass slides that allow rapid initiation of data collection, but
compromise standardization and eliminates features such as automated cell counting offered by the
volumetric chamber. Therefore a need exists to develop microfluidic capabilities to enable rapid mixing
of sperm cells with activation solution during transport into a volumetric viewing chamber for reliable
and accurate assessment by CASA. In addition, microfluidics offers a new field of opportunity for
application with aquatic species gametes.
With the continued development of genomic tools conserved and species-specific molecular mechanisms can
be identified, and comparative studies among vertebrate species are becoming commonplace for human
biomedical research. As the largest class of vertebrates, bony fishes offer almost unlimited versatility
for research. Generally, model fish require features such as small body size, high fecundity, ease of
culture, and most importantly, specific characteristics for particular research topics. Currently, the
most widely used fish models are zebrafish Danio rerio (Driever et al. 1994, Kari et al. 2007), medaka
Oryzias latipes (Wittbrodt et al. 2002), live-bearing fishes of the genus Xiphophorus (Walter and Kazianis
2001), mummichog Fundulus heteroclitus (Atz 1986, Burnett et al. 2007) and pufferfish Fugu rubripes
(Elgar et al. 1996). With extensive studies using these fish models, thousands of specific strains and
lines have been created, and are currently housed worldwide as live animals in resource centers, such as
the Zebrafish International Resource Center (University of Oregon, Eugene) which holds around 1080 inbred,
transgenic, knockout and mutant strains; the University of Georgia (Athens), which holds several inbred
and transgenic medaka lines, and the Xiphophorus Genetic Stock Center
(Texas State University, San Marcos), which holds 61 inbred lines (of which most have been maintained for
50 to more than 100 generations) of 24 species. Preservation of the genetic resources of these and other
valuable fishes presents significant and urgent challenges. Gamete or embryo cryopreservation is a useful
approach to address these challenges.
Ideally, a conservation program should include the preservation of sperm, eggs, embryos and larvae
to secure the revival of species or strains. Currently, cryopreservation techniques in fish are mostly
applied to sperm. Cryopreservation has not been successful for eggs and early embryos because of their
large size, high lipid content, polar organization (Blesbios and Labbe 2003), and membrane impermeability
(Hagedorn et al. 1998). Sperm cryopreservation in fish mostly has focused on large-bodied aquaculture
species, such as salmonids, carps, and catfishes, and only several studies have addressed aquarium fishes.
Due to the small body sizes and limited sperm availability, sperm cryopreservation in aquarium fishes
presents challenges, such as in experimental design, gamete collection, and artificial fertilization,
especially in live-bearing fishes (Tiersch 2001).
We intend for this review to provide an overview of sperm cryopreservation in zebrafish, medaka,
and Xiphophorus, the most important biomedical fish models, and hope it can serve as a template for
research on other aquarium fishes. These three groups possess some distinct differences. For example,
they occur naturally in two habitats: strict freshwater (zebrafish and Xiphophorus) (Hawkins et al. 2001)
and brackish-water-accommodated freshwater (medaka)
(Inoue and Takei 2002), and they have two reproduction modes: external fertilization (zebrafish and medaka)
and internal fertilization (Xiphophorus).
Cryopreservation is a process where biological materials such as cells and tissues are preserved by cooling
to very low temperatures, typically, -196 °C (the boiling point of liquid nitrogen), yet remain viable
after subsequent warming to temperatures above 0 °C. For sperm cryopreservation, this process typically
includes gamete collection, suspension of sperm in an extender, quality assessment, addition of
cryoprotectants, equilibration, freezing, thawing and fertilization, and subsequent development of early
life stages for assessment of cryopreservation success (Tiersch 2000). The term “extender” refers to a
solution of salts, sometimes including organic compounds such as sugars that helps maintain sperm viability
prior to and during the freezing process. This term has also been used to include cryoprotectant
molecules (e.g., dimethyl sulfoxide or methanol) in some literature; however, its use in this chapter
refers to salt solutions only (e.g., calcium-free Hanks’ balanced salt solution).
Numerous studies in sperm cryopreservation have been devoted to optimizing specific components
of cryopreservation procedures. However, aside from those factors mentioned above, other factors such
as sample density, freezing container, starting temperatures, final temperatures (before plunging into
liquid nitrogen), and dilution and cryoprotectant removal after thawing could also affect results
(Leibo 2000). This review calls attention to the importance of the cumulative and interacting
effects arising from all activities in the cryopreservation process. The remainder of this chapter
is intended to provide an overview of sperm cryopreservation in oysters with an emphasis on
identifying problems, variation, and lack of standardization among previous studies. This review
also points out where and how this research differs from previous studies, and the importance of
standardization for the future potential commercialization of cryopreserved sperm in aquatic species.
Genetic improvement of channel catfish Ictalurus punctatus broodstock is needed to maintain desirable characteristics for aquaculture (e.g., growth rate, feed conversion), to avoid problems associated with inbreeding (e.g., reduction of immune system function), and to ensure conservation of genetic resources. Additionally, hatchery techniques are required to increase the cost effectiveness of aquaculture breeding programs. For example, buffered extender solutions have been the used for refrigerated storage and cryopreservation of channel catfish sperm (Guest et al. 1976, Tiersch et al. 1994, Christensen and Tiersch. 1996). These methods can be used for selective breeding to improve commercially important traits. However, the typical farm does not have the resources necessary for the preparation of extenders used for refrigerated storage and cryopreservation of sperm. Simplification of processes, such as the use of minimal extenders, could advance development of breeding programs and improve production economics. To that end, this study evaluated simplified sperm extenders for use in the refrigerated storage and cryopreservation of channel catfish sperm. Our goal was to identify extenders based on readily available food-grade ingredients useful for artificial spawning that would be accessible to a wide range of farmers and breeders. The objectives were to evaluate the effects of extender composition on refrigerated storage of sperm, cryopreservation of sperm, fertilization percentage, and growth of fingerlings.
When dealing with artificial spawning of fish species, gametes must often be stored at specific temperatures
to prolong fertilizing ability. Many studies have dealt with the issue of temperature and storage of
sperm, eggs and embryos (e.g., Rothbard et al. 1996, Dinnyes et al. 1997). Storage of gametes can facilitate
seedstock production and selective breeding, assist preservation of genetic diversity and expand research
opportunities. Refrigeration of sperm also offers several advantages including hybridization and
crossbreeding which can be performed in the hatchery. This particular study was part of a larger evaluation
of the storage of eggs of koi carp Cyprinus carpio at different temperatures (Glenn 1998, Glenn and
Tiersch 2002).
The purpose of this study was to modify general-purpose refrigerators to increase useable space
and reduce spatial temperature variation to provide incubators for storage of gametes. Economics also
played an important role. Commercially available incubators similar to the refrigerators in this study
cost US $1000 or more. Other incubators with precise thermostat settings from 5 to 50 oC cost in the
thousands of dollars. Information about the construction of incubators is limited, but a previous design
was used as a guide for the use of an external hydraulic-action thermostatic controller (Tiersch and
Tiersch 1993, Glenn and Tiersch 2000).
Whether consumers in the United States know it or not, approximately 30% of their seafood is being raised
through aquaculture production (Harvey 2003). Aquaculture in the United States primarily consists of
production of foodfish, ornamental fish, baitfish, mollusks, crustaceans, aquatic plants, and reptiles
such as alligators and turtles (ERS 2003). Rapid growth has occurred within many aquaculture industries
since the 1980s, resulting in a quadrupling of U.S. aquaculture production during that time. In 2005 the
total value of U.S. aquaculture production was approximately $1.09 billion (2005 U.S. Census of Aquaculture).
Foodfish production, which included catfish, trout, salmon, tilapia, hybrid striped bass, sturgeon,
yellow perch, and walleye, made up approximately 60% of total sales (2005 U.S. Census of Aquaculture).
Despite the growth in U.S. aquaculture, a variety of challenges have emerged, for example, in
production of channel catfish Ictalurus punctatus which is an important regional and national market.
In 2007, nearly 500 million pounds were processed in the southeastern U.S., and prices paid to farmers
ranged from $0.65 to $0.83 per pound (USDA NASS 2008). In the past few years, however, the industry has
experienced a catastrophic reduction and continues to face severe challenges. Between 2001 and 2007 the
number of fingerlings produced by the four major states (Mississippi, Arkansas, Alabama, and Louisiana)
shrank by approximately 22% (USDA NASS 2008), and approximately 40,000 water-acres were taken out of production.
The decrease in production is attributable to reduced profit margins from low prices paid to farmers,
increased costs of fuel and feed ingredients, and increased foreign competition. Asian countries have
become the predominant suppliers in global aquaculture markets, with China alone accounting for over 70%
of the total volume of world aquaculture production, and close to 50% of the total world value (FAO 2004).
Less expensive labor and an abundance of land suitable for aquaculture production has led to the emergence
of global competition. Consequently, a major challenge for U.S. aquaculture is to remain competitive in
domestic and global markets. One way to accomplish that is to develop and adopt new technologies and
production systems that allow lower production costs. One approach with essentially unexploited potential
to increase efficiency is to improve the genetic attributes of fish stocks. Greater control over genetics
will allow producers to improve product consistency and lower production costs.
Accordingly, we sought to quantify the awareness and perceived value of the kinds of genetic
improvement that could be made available through commercial-scale use of cryopreservation and development
of markets for genetic resources within U.S. aquaculture. We employed a choice-based conjoint analysis
to determine the importance of genetic improvements to grow-out producers and estimated willingness-to-pay
for selected attributes.
High-throughput cryopreservation has been widely applied in livestock industries such as dairy for decades
(Pickett and Berndtson 1974), and cryopreserved germplasm constitutes an independent industry encompassing
animal breeding, preservation of genetic diversity, and medical research. Fish sperm cryopreservation
was demonstrated at about the same time as for humans and livestock species (Blaxter 1953), however,
despite the large-scale application in mammals, it remains at a research scale for aquatic species.
During the past decades, there have been more than 200 aquatic species reported for sperm cryopreservation
(Tiersch and Mazik 2000) and this number is increasing (although an accurate current estimate is not
available). However, there is currently no system for large-scale production of cryopreserved germplasm
of aquatic species. Recently, a survey of fish culturists revealed a high demand for genetic improvements
of the type that can be provided by cryopreservation (Boever 2006). Therefore, high-throughput
cryopreservation for aquatic species comes with high expectations.
As stated above, high-throughput cryopreservation has been widely applied in livestock industries
for decades. Its development grew out of advances made in the laboratory, and was scaled up for
increased processing speed, capability for mass production, and quality assurance. Available equipment
and processes in place for livestock and biomedical applications have been tested for feasibility of
use with aquatic species (e.g., Haffray et al. 2008), with extensive work for more than 15 yr at the T.
E. Patrick Dairy Improvement Center of the Louisiana State University Agricultural Center. For example,
sperm of blue catfish Ictalurus furcatus was cryopreserved at this commercial facility using procedures
developed for dairy bulls (Lang et al. 2003), as was done for sperm of common carp Cyprinus carpio,
channel catfish Ictalurus punctatus, three species of sturgeon (Scaphirhynchus and Acipenser), striped
bass Morone saxitilis, white bass M. chrysops, yellow bass M. mississippiensis, and marine species such
as spotted seatrout Cynoscion nebulosus, red snapper Lutjanus campechanus (Roppolo 2000), and diploid
and tetraploid Pacific oysters Crassostrea gigas (Dong et al. 2005, 2007a). Since then protocols and
automated equipment developed for mammals have been specifically adapted for use with aquatic species
(described below) including zebrafish Danio rerio (Yang et al. 2007), blue catfish (Hu et al. 2010),
Eastern oyster Crassostrea virginica (Yang et al. unpublished data), channel catfish, and Atlantic
salmon Salmo salar (Hu et al. unpublished data).
With the development of genomic research technologies, comparative genome studies among vertebrate species are becoming commonplace for human biomedical research. Fish offer unlimited versatility for biomedical research. Extensive studies are done using these fish models, yielding tens of thousands of specific strains and lines, and the number is increasing every day. Thus, high-throughput sperm cryopreservation is urgently needed to preserve these genetic resources. Although high-throughput processing has been widely applied for sperm cryopreservation in livestock for decades, application in biomedical model fishes is still in the concept-development stage because of the limited sample volumes and the biological characteristics of fish sperm. High-throughput processing in livestock was developed based on advances made in the laboratory and was scaled up for increased processing speed, capability for mass production, and uniformity and quality assurance. Cryopreserved germplasm combined with high-throughput processing constitutes an independent industry encompassing animal breeding, preservation of genetic diversity, and medical research. Currently, there is no specifically engineered system available for high-throughput of cryopreserved germplasm for aquatic species. This review is to discuss the concepts and needs for high-throughput technology for model fishes, propose approaches for technical development, and overview future directions of this approach.
The goal of this study was to develop a rapid and reliable method of quantifying the gonadal condition of eastern oysters Crassostrea virginica based on a gonad-to-body ratio (GBR). The objectives were to compare a previously established transect method with three alternative computer-based image analysis (IA) methods based on acquisition time, composite GBR values, and GBR values at different stages of gonadal development. The first IA method calculated the area of the gonad and body while eliminating the dorsal and ventral curvature of the gonad, the second calculated the area of the gonad and body while eliminating the gill area, and the third calculated the total area of the gonad and body. The GBR values from the first and third IA methods were not different from those from the transect method. The second IA method resulted in higher GBR values in 80% of the measurements compared with the other three methods. However, measuring the gonadal area and total body area was five times faster than with the transect method and, excluding the curvature, was nine times faster but susceptible to visual error. Based on the speed and comparability with the transect method, the third IA method was the most useful for quantifying gonadal development.
This report describes the feasibility of using vitrification for fish sperm. Vitrification can be used to preserve samples in the field and offers an alternative to conventional cryopreservation, although it has not been systematically studied for sperm of aquatic species. The overall goal of the project was to develop streamlined protocols that could be integrated into a standardized approach for vitrification of aquatic species germplasm. The objectives of the present study in channel catfish (Ictalurus punctatus) were to: (1) evaluate the acute toxicity of 5%, 10%, 20% and 30% methanol, N,N-dimethyl acetamide, dimethyl sulfoxide, 1,2-propanediol, and methyl glycol; (2) evaluate a range of devices commonly used for cryopreservation and vitrification of mammalian sperm; (3) compare vitrification with and without cryoprotectants; (4) evaluate the post-thaw membrane integrity of sperm vitrified in different cryoprotectant solutions, and (5) evaluate the ability of vitrified sperm to fertilize eggs. Cryoprotectant concentrations of higher than 20% were found to be toxic to sperm. Methanol and methyl glycol were the least toxic at a concentration of 20% with an exposure time of less than 5 min. We evaluated a method reported for human sperm, using small volumes in loops (15 microL) or cut standard straws (20 microL) with and without cryoprotectants plunged into liquid nitrogen. Cryoprotectant-free vitrification using loops did not yield fertilization (assessed by neurulation), and the fertilization rates observed in two trials using the cut standard straws were low (~2%). In general, fertilization values for vitrification experiments were low and the use of low concentrations of cryoprotectants yielded lower fertilization (<10%) than the use of vitrification solutions containing high cryoprotectant concentrations (as high as 25%). The highest neurulation obtained was from a mixture of three cryoprotectants (20% methanol + 10% methyl glycol + 10% propanediol) with a single-step addition. This was reflected in the flow cytometry data from which the highest membrane integrity using loops was for 20% methanol + 10% methyl glycol + 10% propanediol (~50%). We report the first successful sperm vitrification in fish and production of offspring from vitrified sperm in channel catfish. Although the fertilization values were low, at present this technique could nevertheless be used to reconstitute lines (especially in small aquarium fishes), but it would require improvement and scaling up before being useful as a production method for large-bodied fishes such as catfish.
Motility activation of fish sperm typically responds to levels of specific ions or osmotic pressure differences between the surrounding water and body tissues. In general, the sperm of marine fishes are activated by an increase in osmotic pressure (hypertonic salinity), and that of freshwater species by a decrease (hypotonic salinity). These stenohaline species exist in relatively stable environments, however, estuarine fishes are exposed to rapidly changing and broad salinity ranges, often resulting in external osmotic pressures that include those of the body (isotonic). To assess the ability of Fundulus grandis sperm to adapt to changes in salinity, adult males were acclimated to salinities of 0, 5, 10, 20, 35, or 50 ppt and held for 30 d. The testes were dissected from the fish and sperm were activated with deionized water, various osmolalities (100–1000 mOsmol/kg) of Hanks' balanced salt solution (HBSS), calcium-free HBSS (Ca2+-Free HBSS), and sodium chloride solution (NaCl). The deionized water did not activate sperm motility regardless of the acclimated salinity. Compared to HBSS, Ca2+-Free HBSS and NaCl activated sperm motility with a significantly lower percentage at the same osmolalities. The osmolality eliciting the highest motility activation was significantly different (P b 0.01) among acclimated groups and shifted from 300 mOsmol/kg (ranging from 200 to 500) for sperm collected from 5 ppt, 500 mOsmol/kg (ranging from 200 to 800) for sperm collected from 10 ppt, 600 mOsmol/kg (ranging from 400 to 700) for sperm collected from 20 ppt, 800 mOsmol/kg (ranging from 200 to 900) for sperm collected from 35 ppt, and 900 mOsmol/kg (ranging from 600 to 1000) for sperm collected from 50 ppt. Motility peaked after 30 s exposure to HBSS, and decreased over 10 min. Motility exhibited a similar initial pattern when exposed to Ca2+-Free HBSS, however, the sperm gained motility at lower osmolalities over 10 min, exhibiting multiple peaks. These results indicate that environmental salinity can significantly influence sperm behavior in adult males of F. grandis with substantial changes after only 30 d of acclimation. As such, this should be considered as a major unrecognized variable in sperm research in this species and can be considered for use in optimizing protocols addressing in-vitro fertilization and cryopreservation. Whether this phenomenon is unique to Fundulus or is a characteristic of euryhaline fishes remains unresolved.
Xiphophorus fishes are well-established models for biomedical research of spontaneous or induced tumors, and their use in research dates back to the 1930s. Currently, 58 well-pedigreed lines exist among 24 Xiphophorus species housed as live animals at the Xiphophorus Genetic Stock Center. The technique of sperm cryopreservation has been applied to preserve these valuable genetic resources, and production of offspring has been reported with cryopreserved sperm in two species (X. helleri and X. couchianus). The goal of this research was to establish protocols for sperm cryopreservation and artificial insemination that yield live young in X. maculatus, a widely used research species. The objectives were to: 1) collect basic biological characteristics of males, and quantify the sperm production yield after crushing of dissected testis; 2) cryopreserve sperm from X. maculatus by adapting as necessary the protocols for sperm cryopreservation of X. helleri and X. couchianus; 3) use cryopreserved sperm to inseminate virgin females of X maculatus and other species (X. helleri and X. couchianus), and 4) compare experimental trials over a 3-year period to identify opportunities for improving female fecundity. In total, 117 males were used in this study with a standard length of 2.5± 0.3 cm (mean±SD), body weight of 0.474± 0.149 g, and dissected testis weight of 7.1 ± 3.7 mg. Calculation of sperm availability showed 5.9 ± 2.8 ×106 sperm cells per mg of testis weight. Offspring were produced from cryopreserved sperm. Male-to-male variation (1–70%) was observed in post-thaw motility despite little variation in motility before freezing (60–90%) or genetic variation (~100 generations of sib-mating). Comparisons of biological factors of males did not have significant correlations with the production of live young, and the influence of females on production of young was identified from the comparison of artificial insemination over 3 years. Overall, this study describes offspring production from cryopreserved sperm in a third species of Xiphophorus fishes, and identifies the opportunities for improving female fecundity which is essential for establishment of germplasm repositories for Xiphophorus fishes.
This study reports the first production of offspring with vitrified sperm from a live-bearing fish Xiphophorus hellerii. The overall goal of this study was to develop streamlined protocols for integration into a standardized approach for vitrification of aquatic species germplasm. The objectives were to (1) estimate acute toxicity of cryoprotectants, (2) evaluate vitrification solutions, (3) compare different thawing methods, (4) evaluate mem-brane integrity of post-thaw sperm vitrified in different cryoprotectants, and (5) evaluate the fertility of vitrified sperm. Nine cryoprotectants and two commercial vitrification additives were tested for acute toxicity and glass forming ability, alone and in combination. Two vitrification solutions, 40% glycerol (Gly) and 20% Gly + 20%ethylene glycol (EG) in 500 mOsmol/kg Hanks’ balanced salt solution (HBSS), were selected for vitrification of 10 lL sperm samples using inoculating loops plunged into liquid nitrogen. Samples were thawed at 24C (one loop in 5 lL of HBSS or three loops in 500 lL of HBSS). Samples thawed in 500 lL were concentrated by centrifugation (1000 g for 5 min at 4'C) into 5 lL for artificial insemination. Offspring were produced from virgin females inseminated with sperm vitrified with 20% Gly + 20% EG and concentrated by centrifugation.
Sperm cryopreservation is a valuable tool in germplasm preservation and breeding. Despite many studies, re-liable and routine cryopreservation of oyster sperm remains a challenge. The goal of this study was to develop a reliable protocol for sperm cryopreservation of the eastern oyster Crassostrea virginica with high-throughput processing by using two types of 0.5-ml straws (French and CBS™ high security straws). The objectives were to: 1) evaluate the effect of 10% methanol, dimethyl sulfoxide (DMSO), and propylene glycol as cryoprotectants at cooling rates of 5, 20 and 40 °C/min from 5 to −80 °C and thawing at 30, 40 and 50 °C; 2) evaluate the effect of cooling rates of 10, 15, 20, 25 and 30 °C/min with 10% DMSO as cryoprotectant and thawing at 40 °C; 3) evaluate the effect of equilibration time (10–60 min) before freezing; 4) evaluate the effect of sperm concentrations from 1×108 to 1×109 for freezing, and 5) verify the established protocol by freezing sperm from 16 individual males. Among the three cryoprotectants, DMSO yielded the highest post-thaw motility at a cooling rate of 20 °C/min when thawed at 30 or 40 °C. Further evaluation of cooling rates of 10, 15, 20, 25 and 30 °C/min showed that 20 or 25 °C/min yielded the highest post-thaw motility (34±5%) and fertility (77±12%) for French straws and CBS straws (28±3% and 69± 14%). Equilibration times of 10 to 60 min did not cause significant differences in post-thaw motility when freezing with 10% DMSO at a cooling rate of 25 °C/min. Also, sperm concentrations ranging from 1×108 to 1×109 at freezing did not cause significant differences in post-thaw motility. Sperm concentration after thawing was not differ-ent compared to that before freezing, and no agglutination was observed in the post-thaw samples. Finally, after thawing, sperm cryopreserved from 16 males with this protocol showed 58±24% fertility (from 18 to 86%) for French straws, and 54±21% fertility for CBS straws (from 18 to 95%). Overall, this study provided a reliable protocol for sperm cryopreservation in the eastern oyster with potential for high-throughput processing which can produce thousands of straws per day with homogenous and reliable quality.
High-throughput cryopreservation operations of fish sperm is a technology being developed by researchers today. This paper first formulates a grouping problem in high-throughput cryopreservation operations of fish sperm and then develops a heuristic and four metaheuristic algorithms for its solution. The heuristic is modified from one originally proposed for the assembly line balancing problem. The four metaheuristic algorithms include simulated annealing (SA), tabu search (TS), ant colony optimization (ACO), and a hybrid differential evolution (hDE). For each metaheuristic algorithm, four different initialization methods were used. For both SA and TS, five different neighborhood solution generation methods were also studied. Real world data collected from a high-throughput cryopreservation operation was used to test the effectiveness of algorithms with different initialization and neighborhood solution generation methods. For comparison, a base line of grouping by processing order was also established. The results indicate that: (i) all algorithms performed better than the base line; (ii) using the result of the modified heuristic as the initial solution of metaheuristic algorithms lead to a better solution; the amount of improvement varied from algorithm to algorithm; (iii) among the five neighborhood solution generation operators, insertion operator was the best; (iv) among all algorithms tested, the hybrid differential evolution is the best, followed by tabu search in terms of average objective value.
Ultrasound imaging analysis involves development of an effective combination of physical properties, equipment, instrument settings, and protocols. This review focuses on the application of ultrasonography to fish reproduction. The goal was to assemble a comprehensive reference data set to serve as a decision-enabling tool for potential users. The specific objectives were to (1) identify the ultrasound equipment, settings, and procedures used during examination, (2) review the fish handling procedures used during examination, and (3) review current data on sex identification and reproduction indices developed using ultrasonography. The 27 studies selected for inclusion in this review represent 21 fish species. Most (96%) of the studies reported the model name for the ultrasound unit, but only 19% reported the probe model. The most reported probe features were the frequency capability (96% of the studies) and array format (linear, sector, or annular; 81%). The majority of the studies (89%) did not report any of the control settings used. The combinations of handling and ultrasound procedures were variable even within the same species, and the majority (78%) of the studies included a form of restraint. None of the studies simultaneously integrated the use of unrestrained, unanesthetized, submersed fish with a submersed waterproof probe, which would enable the use of water as a transmission medium for ultrasound. Size, life stage, gonadal growth of reproductively active adults, and fish morphology influenced the ability to use ultrasonography for sex identification and the development and application of qualitative and quantitative reproductive indices. The utility of ultrasonography in fish reproduction has been repeatedly validated, and innovative indices for noninvasive use have been developed. However, this review identifies a clear lack of consistency in reporting of instrument settings and handling procedures and provides suggestions for standardizing the use of ultrasonography with aquatic species.
This study addressed the development of rapid, straightforward, and minimally stressful procedures for the ultrasound imaging of ovaries of channel catfish Ictalurus punctatus in a commercial hatchery setting. The objectives were to (1) describe the ultrasound imaging equipment and settings used, (2) describe the fish handling procedures during imaging, and (3) illustrate image orientation with respect to the physical positioning of the probe and the catfish. Ultrasound images of the ovaries of channel catfish were recorded as digital video recordings (audio video interleave format) and as still images (ultrasound image format files). This study integrated the use of nonanesthetized, submersed fish within a recirculating tank system or portable container and a submersed waterproof probe, which enabled us to use water as a transmission medium for ultrasound. This allowed us to image the fish in ventral recumbency (upright swimming position) without using a physical restraint in the tank system, or by positioning the fish in the portable container by adjusting the position of its caudal peduncle with one hand and that of the probe with the other hand. The ease of using this technique allows it to be employed as a systematic method for fish handling under laboratory and hatchery conditions. The detailed ultrasound imaging procedures and instrument control settings reported can be used in future testing, improvement, and standardization of procedures for viewing ovaries in channel catfish and potentially other species.
A passive, planar micromixer design based on logarithmic spirals is presented. The device was fabricated using polydimethylsiloxane soft photolithography techniques, and mixing performance was characterized via numerical simulation and fluorescent microscopy. Mixing efficiency initially declined as the Reynolds number increased, and this trend continued until a Reynolds number of 15 where a minimum was reached at 53%. Mixing efficiency then began to increase reaching a maximum mixing efficiency of 86% at Re = 67. Three-dimensional (3D) simulations of fluid mixing in this design were compared to other planar geometries such as the Archimedes spiral and Meandering-S mixers. The implementation of logarithmic curvature offers several unique advantages that enhance mixing, namely a variable cross-sectional area and a logarithmically varying radius of curvature that creates 3D Dean vortices. These flow phenomena were observed in simulations with multilayered fluid folding and validated with confocal microscopy. This design provides improved mixing performance over a broader range of Reynolds numbers than other reported planar mixers, all while avoiding external force fields, more complicated fabrication processes and the introduction of flow obstructions or cavities that may unintentionally affect sensitive or particulate-containing samples. Due to the planar design requiring only single-step lithographic features, this compact geometry could be easily implemented into existing micro-total analysis systems requiring effective rapid mixing.
Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have different evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30uC/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (~5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had~50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (-196'C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems.
Sperm viability in aquatic species is increasingly being evaluated by motility analysis via computer-assisted sperm analysis (CASA) following activation of sperm with manual dilution and mixing by hand. User variation can limit the speed and control over the activation process, preventing consistent motility analysis. This is further complicated by the short interval (i.e., less than 15 s) of burst motility in these species. The objectives of this study were to develop a staggered herringbone microfluidic mixer to: 1) activate small volumes of Danio pearl zebrafish (Danio albolineatus) sperm by rapid mixing with diluent, and 2) position sperm in a viewing chamber for motility evaluation using a standard CASA system. A herringbone micromixer was fabricated in polydimethylsiloxane (PDMS) to yield high quality smooth surfaces. Based on fluorescence microscopy, mixing efficiency exceeding 90% was achieved within 5 s for a range of flow rates (from 50 to 250 microL/h), with a correlation of mixing distances and mixing efficiency. For example, at the nominal flow rate of 100 microL/h, there was a significant difference in mixing efficiency between 3.5 mm (75 +/- 4%; mean +/- SD) and 7 mm (92 +/- 2%; P = 0.002). The PDMS micromixer, integrated with standard volumetric slides, demonstrated activation of fresh zebrafish sperm with reduced user variation, greater control, and without morphologic damage to sperm. Analysis of zebrafish sperm viability by CASA revealed a statistically higher motility rate for activation by micromixing (56 +/- 4%) than manual activation (45 +/- 7%; n = 5, P = 0.011). This micromixer represented a first step in streamlining methods for consistent, rapid assessment of sperm quality for zebrafish and other aquatic species. The capability to rapidly activate sperm and consistently measure motility with CASA using the PDMS micromixer described herein will improve studies of germplasm physiology and cryopreservation.
The use of fluorescent staining and flow cytometry to assess sperm quality in aquatic species has increased over the past decade, but comparisons among studies are difficult or impossible due to variation in application, analysis, and reporting of protocols and data. The goal of the present study was to determine the effect of exposure to two cryoprotectants commonly used for cryopreservation of spermfrom aquatic species on the accuracy of flow cytometric assessment of sperm quality. Membrane integrity of zebrafish (Danio rerio) sperm exposed to 10% and 20% methanol and dimethyl sulfoxide (DMSO) in 300 mOsm kg−1 Hanks' balanced salt solution (HBSS) or calcium-free HBSS was determined using SYBR 14/propidium iodide staining. Both cryoprotectants significantly affected forward-scatter and side-scatter characteristics of sperm samples, resulting in significant changes in the number of total and gated events, and in the number and percentage of intact cells. These results indicate that it cannot be assumed that the approach to flow cytometric analysis of fresh sperm will be applicable to cryoprotectant-treated or cryopreserved sperm. In total, we document examples of five potentially interacting factors that produce errors of 5 to 50% each, resulting in underestimates and overestimates of total and intact sperm (actual numbers and percentages) in the presence of the two most commonly used cryoprotectants at the concentrations used most often for cryopreservation of sperm from aquatic species. This study provides methods to reduce or eliminate these errors and recommendations necessary for standardization and reporting.
Cryopreservation of sperm from Xiphophorus fishes has produced live young in three species: X. hellerii, X. couchianus, and X. maculatus. In this study, the goal was to establish protocols for sperm cryopreservation and artificial insemination to produce live young in X. variatus, and to identify needs for repository development. The objectives were to: 1) collect basic biological characteristics of males; 2) cryopreserve sperm from X. variatus, 3) harvest live young from cryopreserved sperm, and 4) discuss the requirements for establishment of sperm repositories. The 35 males used in this study had a body weight of 0.298 – 0.096 g (mean – SD), body length of 2.5 – 0.2 cm, and testis weight of 6.4 – 3.4 mg. The sperm production per gram of testis was 2.33 – 1.32 · 109 cells. After freezing, the post-thaw motility decreased significantly to 37% – 17% (ranging from 5% to 70%) ( p = 0.000) from 57% – 14% (40%–80%) of fresh sperm (N = 20). Artificial insemination of post-thaw sperm produced confirmed offspring from females of X. hellerii and X. variatus. This research, taken together with previous studies, provides a foundation for development of strategies for sperm repositories of Xiphophorus fishes. This includes: 1) the need for breeding strategies for regeneration of target populations, 2) identification of minimum fertilization capacity of frozen samples, 3) identification of fish numbers necessary for sampling and their genetic relationships, 4) selection of packaging containers for labeling and biosecurity, 5) assurance of quality control and standardization of procedures, 6) information systems that can manage the data associated with cryopreserved samples, including the genetic data, 7) biological data of sampled fish, 8) inventory data associated with frozen samples, and 9) data linking germplasm samples with other related materials such as body tissues or cells saved for DNA and RNA analyses.
A sperm cryopreservation protocol for the Indian major carp, Labeo calbasu, was developed for long-term preservation and artificial fertilization. Milt collected from mature male fish were placed in Alsever’s solution (296 mOsmol kg−1) to immobilize the sperm. Cryoprotectant toxicity was evaluated by motility assessment with dimethyl sulfoxide (DMSO) and methanol at 5, 10 and 15% concentrations. DMSO was more toxic at higher concentrations than methanol, and consequently 15% DMSO was excluded from further study. A one-step cooling protocol (from 5 to 80 ◦C) with two cooling rates (5 and 10 ◦C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia). Based on postthaw motility, the 10 ◦C/min cooling rate with either 10% DMSO or 10% methanol yielded significantly higher (P = 0.011) post-thaw motility than the other rate and cryoprotectant concentrations. Sperm thawed at 40 ◦C for 15 s and fresh sperm were used to fertilize freshly collected L. calbasu eggs and significant differences were observed (P = 0.001) in percent fertilization between cryopreserved and fresh sperm as well as among different sperm-to-egg ratios (P = 0.001). The highest fertilization and hatching rates were observed for thawed sperm at a sperm-to-egg ratio of 4.1 × 10^5 : 1. The cryopreservation protocol developed can facilitate hatchery operations and long-term conservation of genetic resources of L. calbasu.
Sperm cryopreservation isa useful technique for conservation programs aimed at preservation of endangered species and genetic resources in germplasm repositories. Fishes of the genus Xiphophorus provide important biomedical research models, specifically for melanoma studies, and are also used in basic research studies of evolution, toxicology, and endocrinology. Valuable broodstocks and specific Xiphophorus strains or lines have been created and need to be preserved in perpetuity. Although viviparous fishes are valuable research animals, many species are at risk of extinction because of human-induced activities such as water resource overexploitation, pollution, introduction of invasive fish species, and habitat alteration. For fishes with internal fertilization, sperm cryopreservation was essentially unexplored prior ta the recent initiation of research on species of Xiphophorus. Thi:s~review summarizes the current advances of sperm cryopreservation over the past five years within the genus, including the following topics: 1) sperm characteristics and challenges posed for sperm cryopreservation by these unique characteristics; 2) development of a practical protocol for sperm cryopreservation; 3) artificial insemination using cryopreserved sperm for fertility detection, and 4) the outlook for future research. The conclusions in this review may serve as a template for studies on sperm cryopreservation in all viviparous fishes.
Cryopreservation of fish sperm has been studied for decades at a laboratory (research) scale. However, high-throughput cryopreservation of fish sperm has recently been developed to enable industrial-scale production. This study treated blue catfish (Ictalurus furcatus) sperm high-throughput cryopreservation as a manufacturing production line and initiated quality assurance plan development. The main objectives were to identify: (1) the main production quality characteristics; (2) the process features for quality assurance; (3) the internal quality characteristics and their specification designs; (4) the quality control and process capability evaluation methods, and (5) the directions for further improvements and applications. The essential product quality characteristics were identified as fertility-related characteristics. Specification design which established the tolerance levels according to demand and process constraints was performed based on these quality characteristics. Meanwhile, to ensure integrity throughout the process, internal quality characteristics (characteristics at each quality control point within process) that could affect fertility-related quality characteristics were defined with specifications. Due to the process feature of 100% inspection (quality inspection of every fish), a specific calculation method, use of cumulative sum (CUSUM) control charts, was applied to monitor each quality characteristic. An index of overall process evaluation, process capacity, was analyzed based on in-control process and the designed specifications, which further integrates the quality assurance plan. With the established quality assurance plan, the process could operate stably and quality of products would be reliable.
Cryopreservation can preserve genetic materials in perpetuity and can be applied to oyster culture for breeding programs. Protocols exist for sperm cryopreservation in the eastern oyster Crassostrea virginica, but nonlethal sample collection is needed for valuable individuals, such as tetraploids, or specific lines. The goal of this study was to develop nonlethal methods for sperm collection in the eastern oyster. The objectives were (1) to evaluate natural spawning as a collection method, (2) to evaluate anesthesia methods to induce shell opening for biopsy, (3) to evaluate mechanical notching for biopsy, and (4) to verify notching combined with biopsy for collection and cryopreservation. Five males (of 60 oysters) spawned naturally after 7 h, with an average spermconcentration of 1.9 ± 1.03103 cells/mL (in 2 L seawater).No oysters (n¼ 30) responded by opening during 36 h of treatment with 5% Dead Sea salt (containing 33.3% MgCl2), and 22 oysters (of 30) opened during the 36-h treatment with 5% Epson salt (MgSO4). Sperm collected by biopsy had fresh motility of 3%–80% and postthaw motility of 1%–5%; sperm production was 4.53 105 to 2.33108 cells per male.Mechanical notching did not cause mortality to oysters (n = 20). After notching and biopsy with 18-G and 20-G needles, survival was 80% (16 of 20 for each). Sperm production was 5.423107 cells by 18-G needle (n = 8) with fresh motility of 16 ± 12%and postthaw motility of 3 ± 2%, and 1.353108 cells by 20-Gneedle (n¼9)with freshmotility of 21 ± 20%and postthaw motility of 5 ± 4%. No differences were observed between samples biopsied with the 2 needle sizes (P >= 0.074). To verify notching and biopsy for nonlethal sperm collection, a total of 39 oysters were sampled to obtain 20 males, which averaged 99.48 ± 23.17 g total weight, 74.1 ± 6.0 mm shell height, and 60.9 ± 7.2 mm shell length. The sperm production was 3.6 ± 2.13108 cells per male. Biopsied sperm showed 23 ± 12%fresh motility, 13 ± 6% postequilibration motility (after equilibration with 10%of DMSO for 30–60 min before freezing), and 6 ± 4% postthaw motility. Flow cytometry analysis indicated an average of 84 ± 4%of cells with intact plasma membranes for fresh sperm, and 59 ± 9%for postthawsperm. Fertilization by thawed spermaveraged 20 ± 22%(from 1%–87%). No significant differences were observed between the biopsied samples and the dissected samples (lethal collection) for fresh motility (P = 0.550), postequilibration motility (P = 1.000), postthaw motility (P = 0.101), fresh membrane integrity (P = 1.000), or postthawmembrane integrity (P = 1.000), but a difference was observed in fertilization (P = 0.039; biopsied samples, 20 ± 22%; dissected samples, 68 ± 40%). Overall, this study developed notching combined with biopsy for sperm collection and cryopreservation in Eastern oysters that can be applied to valuable individuals and breeding programs.
Cryopreservation is an effective tool for conservation of genetic resources and is becoming increasingly used worldwide with aquatic species. Broadening the application of this technology to a commercial scale through high-throughput approaches has become essential for use with aquatic species. This study addressed high throughput sperm cryopreservation of blue catfish at an industrial level. Our objectives were to: 1) optimize the sperm volume used for thawed sperm; 2) evaluate commercial application of high-throughput cryopreserved sperm with standard hatchery techniques, and 3) initiate evaluation of the fertility relationship between individuals and pooled samples. The results showed that a doubling of the previously established volume did not produce significant improvement in fertilization. The working volume of thawed sperm(2 ml at a concentration of 1 × 109/ml for batches of 100–150 ml channel catfish eggs) was practical. There was no significant difference in fry production after artificial fertilization of 2 million eggs with cryopreserved or fresh sperm. Pooled sperm samples and the individual samples used to form the pools produced similar fertilization rates. Blue catfish sperm is valued as a genetic material for hybrid catfish production, and cryopreservation makes genetic material management possible. This study initiates industrialization of this technology for use with aquatic organisms, and because the technology can be generalized, expands the opportunities for application to other species. High throughput cryopreservation of blue catfish sperm provides new capabilities and can maintain sperm quality sufficiently to support commercial hybrid production.
The eastern oyster Crassostrea virginica can change sex which makes self-fertilization possible if sperm can be cryopreserved. In this study, small (~1 year old) and large (~2–3 years old) oysters were biopsied for sperm collection. Survival of the biopsied oysters after 1 year was 50% for small oysters and 17% for large oysters. Oocytes were collected from sex-reversed females, and self-fertilized with cryopreserved sperm. Of the 24 cryopreserved samples, 14 individuals had ≤1% fertility when crossed with oocytes from unrelated females, indicating that the cryopreserved sperm had reduced fertility. The other 10 individuals had a fertility of 39 25% when crossed with oocytes from unrelated females (nonselfing), but showed a significantly lower success of self-fertilization (12 +/- 16%) (P = 0.008), while aliquots of the same oocytes had a fertilization of 83 +/- 11% when crossing with fresh sperm. Larvae were produced at day 3 in the self-fertilized families (12–94% of the fertilized oocytes), and survived to eyed-larvae stage at days 11–14. Genotyping with 9 microsatellite markers confirmed that the larvae resulted from self-fertilization in four families. This study demonstrated the feasibility of creating selffertilized inbred lines of oysters by use of nonlethal sperm collection and cryopreservation.
Our goal was to develop a standardized approach for sperm vitrification of marine fish that can be applied generally in aquatic species. The objectives were to: (i) estimate acute toxicity of cryoprotectants over a range of concentrations; (ii) evaluate the properties of vitrification solutions (VS); (iii) evaluate different thawing solutions and (iv) evaluate sperm quality after thawing by examination of motility and membrane integrity. Sperm were collected from red snapper (Lutjanus campechanus), spotted seatrout (Cynoscion nebulosus) and red drum (Sciaenops ocellatus). A total of 29 combinations of cryoprotectants were evaluated for toxicity and glass formation. Samples were loaded onto 10-microL polystyrene loops and plunged into liquid nitrogen. There was a significant difference (P < 0.05) in post-thaw motility among VS and among species when using the same VS. The sperm in VS of 15% DMSO + 15% ethylene glycol + 10% glycerol + 1% X-1000TM + 1% Z-1000TM had an average post-thaw motility of 58% and membrane integrity of 19% for spotted seatrout, 38% and 9% for red snapper, and 30% and 19% for red drum. Adaptations by marine fish to higher osmotic pressures could explain the survival in the high cryoprotectant concentrations. Vitrification offers an alternative to conventional cryopreservation.
Emerging commercial-level technology for aquatic sperm cryopreservation has not been modelled by computer simulation. Commercially available software (ARENA, Rockwell Automation, Inc. Milwaukee, WI) was applied to simulate high-throughput sperm cryopreservation of blue catfish (Ictalurus furcatus) based on existing processing capabilities. The goal was to develop a simulation model suitable for production planning and decision making. The objectives were to: (1) predict the maximum output for 8-h workday; (2) analyse the bottlenecks within the process, and (3) estimate operational costs when run for daily maximum output. High-throughput cryopreservation was divided into six major steps modelled with time, resources, and logic structures. The modelled production line processed 18 fish and produced 1164 +/- 33 (mean +/- SD) 0.5-mL straws containing one billion cryopreserved sperm. Two such production lines could support all hybrid catfish production in the United States and 15 such lines could support the entire channel catfish industry if it were to adopt artificial spawning techniques. Evaluations were made to improve efficiency, such as increasing scale, optimizing resources, and eliminating underutilized equipment. This model can serve as a template for other aquatic species and assist decision making in industrial application of aquatic germplasm in aquaculture, stock enhancement, conservation and biomedical model fish.
Sperm cell activation plays a critical role in a range of biological and engineering processes, from fertilization to cryopreservation protocol evaluation. Across a range of species, ionic and osmotic effects have been discovered that lead to activation. Sperm cells of zebrafish (Danio rerio) initiate motility in a hypoosmotic environment. In this study, we employ a microfluidic mixer for the purpose of rapidly diluting the extracellular medium to initiate the onset of cell motility. The use of a microchannel offers a rapid and reproducible mixing profile throughout the device. This greatly reduces variability from trial to trial relative to the current methods of analysis. Coupling these experiments with numerical simulations, we were able to investigate the dynamics of intracellular osmolality as each cell moves along its path through the micromixer. Our results suggest that intracellular osmolality, and hence intracellular ion concentration, only slightly decreases, contrary to the common thought that larger changes in these parameters are required for activation. Utilizing this framework, microfluidics for controlled extracellular environments and associated numerical modeling, has practical applicability in standardizing high-throughput aquatic sperm activation, and more fundamentally, investigations of the intracellular environment leading to motility.
The goal of this study was to evaluate plasma membrane integrity and motility for zebrafish sperm quality assessment along the cryopreservation pathway—from sample collection through refrigerated storage, cryoprotectant equilibration, freezing, thawing, and fertilization. The objectives were to: (1) evaluate the effects of osmolality, extender, and refrigerated storage on sperm plasma membrane integrity and motility, and (2) compare cryopreservation of sperm from farm-raised and well-characterized research populations by evaluating motility and membrane integrity of fresh, post-equilibration (before freezing) and post-thaw sperm, and post-thaw fertility. Osmolality, extender, and storage time each influenced sperm motility and membrane integrity. Isotonic osmolality showed the best protection for motility and membrane integrity compared to hypotonic and hypertonic osmolalities. Of the four tested extenders, Hanks’ balanced salt solution (HBSS) and Ca2+-free HBSS showed the best protection compared with NaCl and glucose, and sperm retained motility and membrane integrity for 24 h of refrigerated storage. Sperm cryopreservation of zebrafish from a farm population (n = 20) and an AB research line (n = 20) showed significant differences in post-thaw fertility (32% +/- 18% vs. 73% +/- 21%). No differences were found in post-thaw motility, although the farm-raised zebrafish possessed a larger body size, testis weight, and higher fresh motility. Correlation analysis of pooled data did not identify correlations among motility, flow cytometry analysis of membrane integrity and recognizable cells, and post-thaw sperm fertility ( p >= 0.202). More research is needed to standardize the fertilization conditions especially sperm-to-egg ratio to avoid possible overabundance of sperm to obscure the differences.
Cryopreservation in aquatic species in general has been constrained to research activities for more than 60 years. Although the need for application and commercialisation pathways has become clear, the lack of comprehensive quality assurance and quality control programs has impeded the progress of the field, delaying the establishment of germplasm repositories and commercial-scale applications. In this review we focus on the opportunities for standardisation in the practices involved in the four main stages of the cryopreservation process: (1) source, housing and conditioning of fish; (2) sample collection and preparation; (3) freezing and cryogenic storage of samples; and (4) egg collection and use of thawed sperm samples. In addition, we introduce some key factors that would assist the transition to commercial-scale, high-throughput application.
Control of sperm concentration is required to ensure consistent and reproducible results for cryopreservation and in vitro fertilization protocols. Determination of sperm concentration is traditionally performed with a counting chamber (e.g., hemocytometer), or more recently with a spectrophotometer. For small-sized biomedical model fishes, the availability of sperm sample is limited to microliters, so it is desirable to develop fast and accurate approaches for concentration determination that also minimize sample use. In this study, a new approach was developed for sperm concentration determination using a flow cytometer (Accuri C6, BD Biosciences, San Jose, CA) with simultaneous measurement of sperm membrane integrity after fluorescent staining with SYBRVR214 and propidium iodide (PI) in sperm from Zebrafish Danio rerio. The goal was to develop a protocol for simultaneous determination of sperm quality and quantity by flow cytometry. The objectives were to (1) determine the effects of sample volume (250 and 500 ml) and analysis volume (10 and 50 ml) on the accuracy of particle counting using standard volumetric validation beads; (2) identify the effective range of sperm concentrations that flow cytometry can measure; (3) test the precision and reproducibility of the sperm concentration measurements; and (4) verify the flow cytometry approach by comparison with measurement with a hemocytometer and a microspectrophotometer. Sample volumes of 250 and 500 ml and analysis volumes of 10 and 50 ml did not affect bead count with the factory-set flow rates of “medium” or “fast,” and the precision and accuracy was retained across a concentration range of 1 x 10^3 - 1 x 10^7 cells/ml. The approach developed in this study was comparable to traditional methodologies such as hemocytometer or microspectrophotometer. This study provides an efficient, accurate, and rapid method for determination of sperm concentration using flow cytometry while providing simultaneous assessment of sperm membrane integrity. Such approaches can reduce the time needed for quantity assessment and maximize the use of valuable sperm samples.
The Southern flounder, Paralichthys lethostigma, is a valuable aquaculture fish with established markets in the USA. All-female production in this species is an important technology for aquaculture because the females usually have body sizes twice those of males at the same age, and sex-reversed males (genotypic XX neomales) are used for all-female production by crossing with genetically normal females. However, sperm volume from the neomales is usually small (<0.5 mL) and limits their application for all-female fish production. Cryopreservation of sperm from these sex-reversed neomales will provide access on demand with increased efficiency to extend the application of neomales. The goal of this study was to develop a protocol for cryopreservation of sperm from the Southern flounder by using an automated high-throughput processing system. The objectives were to: (1) determine the effect of osmolality on activation of sperm motility; (2) evaluate the effect of extender solutions on sperm motility capacity; (3) evaluate the acute toxicity of cryoprotectants (dimethyl sulfoxide [DMSO], propylene glycol, and polyethylene glycol) on sperm motility, and (4) estimate the effect of cooling rate on sperm cryopreservation and post-thaw fertilization. Sperm motility was activated when osmolality was 400 mOsmol/kg or higher. Of the three extender buffers tested, HEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at 300 mOsmol/kg resulted in better protection for sperm motility than did Hanks’ balanced salt solution and Mounib solution at 300 mOsmol/kg during 7 d of refrigerated storage. After 30min equilibration with the cryoprotectant of 15% DMSO, sperm motility was 24±21% (fresh sperm motility without any cryoprotectants was 42%). After cooling at a rate of 20 C/min, post-thaw sperm motility was 8±5% and fertilization was 63±40% evaluated at the 32–64 cell stage (5×10^5 sperm per egg). Overall, a protocol was developed for sperm cryopreservation in the Southern flounder with high-throughput processing, which provides a tool to preserve the valuable genetic resources from neomale flounders, and enables germplasm repository development for the Southern flounder. 1 Correspondence
Fluorescent dyes that binds irreversibly to cellular amines, come in several available emission spectra, and do not poses health concernswere used to evaluatemembrane integrity in fish spermcells. The objectives of the present study were to determine: (1) a working dye concentration for fish sperm samples, and (2) if the traditional propidium iodide/SYBR-14 staining combination was comparable with the amine reactive dye (ARD) methods at identifying cell populationswith intact and compromisedmembranes after spermactivation, refrigerated storage, and exposure to cryoprotectant and surfactant. Zebrafish (Danio rerio) sperm were obtained by stripping, and pooled samples (in triplicate) were used in all tests. Six dilutions of the amine dye (ranging from 0.625 to 0.02 μl/ml) were evaluated, and compared with the traditional staining protocol. A concentration of 0.5 μl/ml ARD was selected to be used in subsequent assays. Sperm suspensions were activated with deionized water to simulate urine contamination. After 10 s, osmolality was increased to stop activation, and the procedure was repeated in 10-s intervals until the sperm remained activated for 120 consecutive sec; membrane integrity was analyzed at each time interval. For the storage assay, sperm suspensions were prepared in Hanks' balanced salt solution at 302 mOsm/kg osmolality (HBSS302), HBSS354 and HBSS402, and evaluated every 2 h for 8 h, and every 24 h for 72 h. Cryoprotectant toxicitywas tested by diluting sperm suspensions in HBSS340 with methanol at 5, 10 and 15% final concentrations. Surfactant toxicity was tested by diluting sperm suspensions in HBSS354 with Triton X-100 at 0.2, 0.15 and 0.1 mM final concentrations. In each toxicity assay, membrane integrity was tested every 20 min for 80 min. The number of membrane-intact cells significantly decreased across time in all treatments (P < 0.05). Significant differences between staining protocolswere observed after activation and after exposure tomethanol at 10 and 15%, and to Triton X-100 (P < 0.05). The average difference, however, was minor (between 1 and 6% in average) in relation to the typical values used for decisionmaking based on this assay. Results showed that thismethod has the potential to contribute greatly to the standardization of cryopreservation in aquatic species. Statement of relevance: QA-QC for repository development in aquatic species.
Commercial production of hybrid catfish (female channel catfish Ictalurus punctatus × male blue catfish Ictalurus furcatus) is reliant on interdependent biological, environmental, and technical procedures. This study addresses one of these critical components – the evaluation of channel catfish ovarian development based on sonographic analysis. The objectives were to: (1) develop a channel catfish ovarian ultrasonography index, (2) test the effect of the spawning trials on fertilization estimates of fish assessed using the ultrasonography index, and (3) evaluate the expected (hypothesized) and observed outcome of the ultrasonography assessments. Seven ovarian morphology classifications were developed based on ultrasonography of 915 channel catfish (N =915 images), and 210 females were selected for spawning. The predictions based on the classifications prior to hormone injection showed a significant effect (P<0.002) on the observed outcomes (viable or nonviable eggs). The probability of correct classification was 0.86–0.89 for Categories 3 (developing), 4 (advanced), and 5 (mature), and 0.93–1.0 for Categories 1 (undeveloped), 2 (underdeveloped), 6 (spawned), and 7 (atretic). The ultrasonography index covered the full range of ovarian dynamics (i.e., recrudescence through spawning). It provided an unobtrusive, direct method of ovarian assessment to work toward improving the efficiency of broodstock selection.
This review is focused on the applications of genome cryobanking of aquatic species including freshwater and marine fish, as well as invertebrates. It also reviews the latest advances in cryobanking of model species, widely used by the scientific community worldwide, because of their applications in several fields. The state of the art of cryopreservation of different cellular types (sperm, oocytes, embryos, somatic cells and primordial germ cells or early spermatogonia) is discussed focusing on the advantages and disadvantages of each procedure according to different applications. A special review on the need of standardization of protocols has also been carried out. In summary, this comprehensive review provides information on the practical details of applications of genome cryobanking in a range of aquatic species worldwide, including the cryobanks established in Europe, USA, Brazil, Australia and NewZealand, the species and type of cells that constitute these banks and the utilization of the samples preserved. Statement of relevance: This review compiles the last advances on germplasmcryobanking of freshwater and marine fish species and invertebrates,with high value for commercial aquaculture or conservation. It is reviewed the most promising cryopreservation protocols for different cell types, embryos and larvae that could be applied in programs for genetic improvement, broodstock management or conservation of stocks to guarantee culture production.
Development of devices through design, prototyping, testing, and fabrication is especially necessary for enhancement of research and eventual application in cryobiology. The advent of 3-dimensional printing offers unique opportunities for this process, given that the materials involved are suitable for use in cryogenic temperatures. We report herein that 3-D printing with polylactic acid (PLA) thermoplastic is ideally suited for cryobiology device development. Devices that are designed and standardized in opensource fashion can be electronically distributed and created locally on increasingly affordable 3-D printers, and can accelerate cryobiology findings and improve reproducibility of results.
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.
Cryopreservation has become an important and accepted tool for long-term germplasm conservation of animals and plants. To protect genetic resources, repositories have been developed with national and international cooperation. For a repository to be effective, the genetic material submitted must be of good quality and comparable to other submissions. However, due to a variety of reasons, including constraints in knowledge and available resources, cryopreservation methods for aquatic species vary widely across user groups which reduces reproducibility and weakens quality control. Herein we describe a standardizable freezing device produced using 3-dimensional (3-D) printing and introduce the concept of network sharing to achieve aggregate high-throughput cryopreservation for aquatic species. The objectives were to: 1) adapt widely available polystyrene foam products that would be inexpensive, portable, and provide adequate work space; 2) develop a design suitable for 3-D printing that could provide multiple configurations, be inexpensive, and easy to use, and 3) evaluate various configurations to attain freezing rates suitable for various common cryopreservation containers. Through this approach, identical components can be accessed globally, and we demonstrated that 3-D printers can be used to fabricate parts for standardizable freezing devices yielding relevant and reproducible cooling rates across users. With standardized devices for freezing, methods and samples can harmonize into an aggregated high-throughput pathway not currently available for aquatic species repository development.
Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, highdensity and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries. Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.
Ultrarapid cooling under the appropriate conditions will produce vitrification, a glass-like state used to cryopreserve small sample volumes, but there are a number of major technical drawbacks impeding the application of vitrification to germplasm of aquatic species. These include a lack of suitable devices, and poor reproducibility and comparability among studies due to a lack of standardization. We used 3-D printing to produce a viewing pedestal coupled with a classification system to rapidly assess frozen film quality of vitrification loops. Classification time declined with practice from 2.1 ± 0.3 sec (mean ± SD) to 1.5 ± 0.2 sec (after 200 assessments), and assessments were consistently made in < 2.5 sec. Classifications should be reported with representative images allowing harmonization for quality control. This approach permits rapid classification and can be applied for the development of methods including the evaluation of vitrification solution components, concentrations of solutions and target cells, and configurations and volumes of new devices. Future studies should address the custom fabrication of 3-D printed vitrification devices for use with aquatic species and other applications.
Quality control (QC) is essential for reproducible and efficient functioning of germplasm repositories. However, many biomedical fish models present significant QC challenges due to small body sizes (<5 cm) and miniscule sperm volumes (<5 microL). Using minimal volumes of sperm, we used Zebrafish to evaluate common QC endpoints as surrogates for fertilization success along sequential steps of cryopreservation. First, concentrations of calibration bead suspensions were evaluated with a Makler counting chamber by using different sample volumes and mixing methods. For sperm analysis, samples were initially diluted at a 1:30 ratio with Hanks’ balanced salt solution (HBSS). Motility was evaluated by using different ratios of sperm and activation medium, and membrane integrity was analyzed with flow cytometry at different concentrations. Concentration and sperm motility could be confidently estimated by using volumes as small as 1 microL, whereas membrane integrity required a minimum of 2 microL (at 1 · 10^6 cells/mL). Thus, <5 microL of sperm suspension (after dilution to 30–150 lL with HBSS) was required to evaluate sperm quality by using three endpoints. Sperm quality assessment using a combination of complementary endpoints enhances QC efforts during cryopreservation, increasing reliability and reproducibility, and reducing waste of time and resources.
Standardized evaluation of sperm quality is essential for research, commercial-scale cryopreservation, and induced spawning. However, standardized methods for evaluation of sperm bundles (spermatozeugmata or spermatophores) have not been established. The purpose of the present study was to use Redtail Splitfin (Xenotoca eiseni) as a model for freshwater livebearing fishes to establish initial standardized methods to collect sperm bundles, and quantitatively and qualitatively evaluate quality-related attributes. No sperm or sperm bundles were able to be collected by stripping. Testes were removed, rinsed, weighed, placed in 50 microL of buffer solution on a glass slide, and crushed gently 3e5 times with angled spade-tip forceps. Sperm bundles were released into the buffer solution and collected with a pipette into 1.5-mL centrifuge tubes. To quantify size and shape, images of bundles were captured with a CCD camera connected to a microscope, and measured with computer software. There was no significant correlation between body wet weight and major bundle axis length (P = 0.6759), minor axis length (P = 0.5658), average axis length (P = 0.5869), aspect ratio (P = 0.7839), and observed area (P = 0.5727). The concentrations of sperm bundles, estimated with the three methods (Makler® counting chamber, a hemocytometer, and direct counting) were significantly different (P < 0.0001). Hemocytometers were suitable for estimation of bundles from X. eiseni. To evaluate activation of sperm, bundles were viewed with a microscope, and classified into one of five phases by evaluating morphology of the bundles and motion of sperm within the bundles as Phase 0 through Phase 4 that represented early through late activation stages. The frequencies and duration of each activation phase were used to evaluate dissociation of sperm bundles and motility capability of spermwithin the bundles. Within 180 min of activation, all five phases were observed. Overall, this study for the first time established standardized methods to collect and evaluate quality-related attributes of sperm bundles. These standardized evaluations provide a basis for further modification, standardization, and generalization, which are useful in research on livebearing fishes involving male gametes, such as studies on cryopreservation, artificial insemination, and in development of germplasm repositories for imperiled species including goodeids.
Crappie, Pomoxis spp., are popular game fish throughout North America and are produced by public and private hatcheries. However, production is limited by a lack of information on tank culture and induced spawning methods. Development of techniques for storage of sperm and in vitro fertilization would increase flexibility in spawning. Therefore, techniques for sperm cryopreservation were examined in white crappie, Pomoxis annularis. Sperm from adult wild white crappie were used to evaluate sperm extender, cryoprotectant agent and concentration, and cooling technique based on post-thaw sperm motility. Percent egg fertilization was also compared between sperm stored in the two best cryopreservation protocols and two different osmotic activator solutions. Sperm were cryopreserved using treatment combinations of two extenders (350 mOsmol/kg Hanks’ balanced salt solution [HBSS] and 350 mOsmol/kg Ca2+free HBSS) and two cryoprotectants (dimethyl sulfoxide [DMSO] and methanol) at concentrations of 5, 10, and 15% that were cooled at four different rates: 5, 10, 20, and 40'C/min. Post-thaw sperm motility and fertilization rates indicated white crappie sperm can be cryopreserved using either extender, cryoprotectants of either 5% DMSO or 10% methanol, and cooling at 40'C/min. A follow-up experiment demonstrated sperm in suspensions on ice retained viability after overnight transport.
Sperm cryopreservation is an essential tool for long-term storage of genetic resources for aquaculture fishes. The goal of this study was to develop an efficient and streamlined protocol for high-throughput processing for sperm cryopreservation in Atlantic salmon, Salmo salar. The objectives were to evaluate: (1) osmolality of blood serum for determining extender osmolality, (2) effects of extenders for fresh sperm dilution and refrigerated storage, (3) effects ofmethanol and dimethyl sulfoxide (DMSO) on fresh sperm motility, and (4) motility and fertility after thawing. In this study, sperm samples were collected at a hatchery site in Canada and shipped to a freezing site located 2200 miles (3550 km) away in the USA. Evaluation of three extenders indicated that Mounib solution was suitable for diluting dry sperm for sample processing. Ten percent of methanol or DMSO was less toxic to sperm cells than was 15% within 30 min. Further testing with methanol at 5, 10, and 15%, and sperm solution : extender dilutions (v:v) of 1:1, 1:3, and 1:19 (at concentrations of 5×10^7, 3×10^8, and 1×10^9 cells/mL) indicated that methanol at 5 and 10% showed less toxicity to fresh sperm within 1 h at sperm:extender dilutions of 1:1 and 1:3. Post-thaw motility of sperm cryopreserved with 10% methanol was significantly higher than that with 10% DMSO, and fertility reflected those results (0–1% in DMSO vs. 38–55% in methanol). Further evaluation of sperm cryopreservation with 10 and 15% methanol at sperm dilution ratios of 1:1, 1:3, and 1:19 indicated that post-thaw motility in 10% methanol was significantly higher than that in 15% methanol, and post-thaw fertility in 10% methanol at 1:1 and 1:3 dilution ratios had fertilization rates similar to that of fresh sperm controls. Sperm samples from 12 males cryopreserved with 10% methanol showed male-to-male variation in post-thaw motility (0–36%). Overall, a simplified standard protocol was established for cryopreservation of shipped sperm of Atlantic salmon using extender without egg yolk and yielded satisfactory post-thaw motility and fertilization rates. This procedure can be readily adopted by aquaculture facilities to take advantage of high-throughput cryopreservation capabilities at remote service centers. Most importantly, this approach lays the groundwork for an alternative commercial model for commercial-scale production, quality control, and development of industrial standards. Control of male variability and sperm quality remain important considerations for future work.
Knowledge of sperm motility activation for viviparous fishes has been limited to study of several species in Poeciliidae, and the dissociation of sperm bundles is even less understood. The goal of this study was to use the endangered Redtail Splitfin (Xenotoca eiseni) as a model to investigate the activation of sperm from viviparous fishes by study of free sperm and spermatozeugmata (unencapsulated sperm bundles). The specific objectives were to evaluate the effects of: (1) osmotic pressure and refrigerated storage (4 °C) on activation of free sperm, (2) osmotic pressure, ions, and pH on dissociation of spermatozeugmata, and (3) CaCl2 concentration and pH on sperm membrane integrity. Free sperm were activated in Ca2+-free Hanks' balanced salt solution at 81–516 mOsmol/kg. The highest motility (19 ± 6%) was at 305 mOsmol/kg and swim remained for 84 h. Glucose (300–700 mOsmol/kg), NaCl (50–600 mOsmol/kg), and KCl, MgCl2, and MnCl2 at 5–160mM activated sperm within spermatozeugmata, but did not dissociate spermatozeugmata. CaCl2 at 5–160mM dissociated spermatozeugmata within 10 min. Solutions of NaCl-NaOH at pH 11.6 to 12.4 dissociated spermatozeugmata within 1 min. The percentage of viable cells had no significant differences (P=0.2033) among different concentrations of CaCl2, but it was lower (P < 0.0001) at pH 12.5 than at pH between 7.0 and 12.0. Overall, this study provided a foundation for quality evaluation of sperm and spermatozeugmata from livebearing fishes, and for development of germplasm repositories for imperiled goodeids.
More than half of fishes in the family Goodeidae are considered to be endangered, threatened, or vulnerable. Sperm cryopreservation is an effective tool for conserving genetic resources of imperiled populations, but development of protocols with livebearing fishes faces numerous challenges including the natural packaging of sperm into bundles. In this study the cryopreservation of sperm bundles (spermatozeugmata) of three goodeids species was evaluated. Sperm quality was evaluated by activation with NaCl-NaOH solution (at 300 mOsmol/kg and pH 11.8), and analysis of dissociable bundles and dissociation duration. Using Redtail Splitfin (Xenotoca eiseni) as a model, the effects of cryoprotectants (dimethyl sulfoxide, methanol, and glycerol) with different concentrations (5–15% v/v %), equilibration exposure times (1–60 min), cooling rates (5–40 °C/min), concentrations (4×10^4–4×10^6 bundles/ml), buffers (HBSS, PBS and NaCl), and buffer osmolalities (200–400 mOsmol/kg) were investigated. After cooling and thawing, sperm bundles maintained their packed form. A specific protocol was developed (10% dimethyl sulfoxide, 20-min equilibration, 10 °C/min cooling rate, 4×106 bundles/ml, and 300 mOsmol/kg HBSS). This protocol yielded 89 ± 5% of post-thaw dissociable bundles with 209 ± 10 s of dissociation duration for X. eiseni, 96 ± 9% with 814 ± 14 s for Blackfin Goodea (Goodea atripinni), and 66 ± 2% with 726 ± 25 s for Striped Goodeid (Ataeniobius toweri). This is the first study of cryopreservation of sperm within bundles for livebearing fishes and provides a basis for establishment of germplasm repositories for goodeids and other livebearers.
Although aquatic species cryopreservation protocols have been studied around the world over the past 60 yr., germplasm repository development efforts and commercialization have begun only recently. The goal of this project was to develop a self-contained mobile laboratory for on-site high-throughput cryopreservation of aquatic species. The objectives of this study were to: (1) identify how a mobile laboratory would function in different operational scenarios, (2) customize an enclosed cargo trailer to function as a mobile laboratory, (3) evaluate the laboratory layout and ability of cryopreservation equipment to operate from generator power, and (4) document the investment costs for private and public groups to integrate a mobile laboratory into an existing cryopreservation facility at three levels of automation and estimate the total cost per trip based on hypothetical assumptions for two scenarios (aquaculture production and repository development). There were three operational designs identified for the mobile laboratory: (1) self-contained work inside the unit using generator power, (2) work inside the unit using external facility power, and (3) using the equipment inside of a host facility. The investment costs for a base-level mobile laboratory ranged between US$5670 and US$5787 for private groups and between US$5208 and US$5315 for public groups. With the addition of a range of automated processing equipment, total investment costs ranged from US$13,616 to US$103,529 for private groups and US$12,494 to US$94,891 for public groups. The total cost per trip to cryopreserve sperm of 59 blue catfish, Ictalurus furcatus, males to produce 6300 0.5-mL French straws was estimated to range fromUS$6089 to US$14,633 for private and between US$5703 and US$16,938 for public groups depending on the level of automation. Total cost per trip to cryopreserve sperm of 500 males of five different species in the genus Xiphophorus to produce 641 0.25-mL French straws was estimated to range from US$6653 to US$7640 for private and US$7582 to US$8088 for public groups depending on level of automation. Overall, a commercial-scale mobile laboratory was developed that can assist current germplasm activities and support future repository and industry development, and the layout information provided can help others to design and build comparable units.
Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservationmethods have produced variable outcomes and low postthaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation,we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process.We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15'C/min. Test thaws from this method produced average motility of 20% – 13% and an average post-thaw fertilization rate of 68% – 16%.
For the past six decades a repeated cycle of developing new cryopreservation protocols or simply reinventing them to counteract a lack of reproducibility has led to hundreds of published studies that have offered little to the establishment of a genetic resources community for aquatic species. This has hampered repository development and inhibited industrial application. Most protocols were developed without standardized approaches, leading to irreproducible studies and questionable or meaningless comparisons. Thus cryopreservation of germplasm in aquatic species would greatly benefit from strategies to facilitate reproducibility. Our objectives were to: (1) identify major sources of irreproducibility across research, small-scale, repository, and commercial-scale development levels; (2) provide recommendations to address reproducibility challenges; and (3) offer suggestions on how researchers can directly influence commercial development and application of cryopreservation research. Sources of irreproducibility include lack of standardized procedural approaches, lack of standardized terminology, and lack of reporting guidelines. To address these challenges, we propose implementation of standard operating procedures, support of stock centers and internet content for development of training programs, and strengthening of the role of scientific journals and reviewers in reducing the frequency of irreproducible outcomes. Reproducibility is the foundation for quality management programs and product reliability, and therefore, standardization is necessary to assure efficient transition to commercial-scale application and repository development. Progress can only be possible through community-based approaches focused on coalescence and consensus of disparate groups involved in aquatic species cryopreservation and management of genetic resources.
Previous studies of sperm cryopreservation of livebearing fish have been limited to two genera within the family Poeciliidae. The goal of the present study was to investigate the feasibility to produce live young of livebearing goodeids (family Goodeidae) with cryopreserved sperm, using aquarium-trade populations of the endangered species Redtail Splitfin (Xenotoca eiseni, Rutter, 1896). Reproductive condition of females was evaluated by histological categorization of ovarian development. A total of 117 females were inseminated with cryopreserved sperm, 81 were inseminated with fresh sperm, 27 were mixed with males for natural breeding, and 30 were maintained without males or insemination. Histological images of 34 mature females indicated 68% of ovaries had primary- or secondary-growth oocytes, and 32% had ovulated eggs. Ovarian development had no significant relationship (P= 0.508) with body wet weight, but had a relationship (P < 0.001) with ovary weight and gonadosomatic index. Sperm cells were observed within ovaries that were fixed at 12 h after insemination with fresh sperm. A total of 29 live young were produced from two females inseminated with thawed sperm (8% post-thaw motility with HBSS300 as extender, 20 min incubation in 15% DMSO, cooling rate at 10 °C/min, and thawing at 40 °C for 7 s), 12 were produced from two females with fresh sperm (1%–20% motility), 41 were produced from five naturally spawned females, and no live young were produced from the female-only group. This study provides a foundation for establishment of germplasm repositories for endangered goodeids to assist conservation programs.
Mechanisms regulating sperm motility activation are generally known in oviparous fishes, but are poorly understood in viviparous species. The mechanism of osmotic-shock induced signaling for oviparous fishes is not suitable for viviparous fishes which activate sperm motility within an isotonic environment. In addition, the presence of sperm bundles in viviparous fishes further complicates study of sperm activation mechanisms. The goal of this study was to establish methodologies to detect intracellular Ca2+ signals from sperm cells within bundles, and to investigate the signaling mechanism of sperm activation of viviparous fish using Redtail Splitfin (Xenotoca eiseni) as a model. Motility was assessed by classification of bundle dissociation and computerassisted sperm analysis, and intracellular Ca2+ was assessed using the fluorescent probe Fura-2 AM. Bundle dissociation and sperm motility increased with extracellular Ca2+ and pH levels. Intracellular Ca2+ signals were detected from sperm within bundles, and increased significantly with extracellular Ca2+ and pH levels. Major channel blockers known to inhibit Ca2+ influx (NiCl2, ruthenium red, GdCl3, SKF-96365, nimodipine, verapamil, methoxyverapamil, mibefradil, NNC 55–0396, ω-Conotoxin MVIIC, bepridil, and 2-APB) failed to inhibit Ca2+ influx, except for CdCl2, which partially inhibited the influx. We propose a novel mechanism for motility regulation of fish sperm: an alkaline environment in the female reproductive tract opens Ca2+ channels in the sperm plasma membrane without osmotic shock, and the Ca2+ influx functions as a second messenger to activate motor proteins controlling flagella movement.
The genetic resources of aquatic biomedical model organisms are the products of millions of years of evolution, decades of scientific development, and hundreds of millions of dollars of research funding investment. Genetic resources (e.g., specific alleles, transgenes, or combinations) of each model organism can be considered a form of scientific wealth that can be accumulated and exchanged, typically in the form of live animals or germplasm. Large-scale maintenance of live aquatic organisms that carry these genetic resources is inefficient, costly, and risky. In situ maintenance may be substantially enhanced and backed up by combining cryopreserved germplasm repositories and genetic information systems with live animal culture. Unfortunately, cryopreservation has not advanced much beyond the status of an exploratory research for most aquatic species, lacks widespread application, and methods for successful cryopreservation remain poorly defined. For most aquatic species biological materials other than sperm or somatic cells are not comprehensively banked to represent and preserve a broad range of genetic diversity for each species. Therefore, new approaches and standardization are needed for repository-level application to ensure reproducible recovery of cryopreserved materials. Additionally, development of new technologies is needed to address preservation of novel biological materials, such as eggs and embryos of aquatic species. To address these goals, the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health (NIH) hosted the Cryopreservation of Aquatic Biomedical Models Workshop on January 7 to 8, 2017, in conjunction with the 8th Aquatic Animal Models of Human Disease Conference in Birmingham, Alabama. The goals of the workshop were to assess the status of germplasm cryopreservation in various biomedical aquatic models and allow representatives of the scientific community to develop and prioritize a consensus of specific actionable recommendations that will move the field of cryopreservation of aquatic resources forward. This workshop included sessions devoted to new approaches for cryopreservation of aquatic species, discussion of current efforts and approaches in preservation of aquatic model germplasm, consideration of needs for standardization of methods to support reproducibility, and enhancement of repository development by establishment of scalable high-throughput technologies. The following three broad recommendations were forwarded from workshop attendees: 1: Establish a comprehensive, centralized unit (“hub”) to programmatically develop training for and documentation of cryopreservation methods for aquatic model systems. This would include development of speciesspecific protocols and approaches, outreach programs, community development and standardization, freezing services and training of the next generation of experts in aquatic cryopreservation. 2: Provide mechanisms to support innovative technical advancements that will increase the reliability, reproducibility, simplicity, throughput, and efficiency of the cryopreservation process, including vitrification and pipelines for sperm, oocytes, eggs, embryos, larvae, stem cells, and somatic cells of all aquatic species. This recommendation encompasses basic cryopreservation knowledge and engineering technology, such as microfluidics and automated processing technologies. 3: Implement mechanisms that allow the various aquatic model stock centers to increase their planning, personnel, ability to secure genetic resources and to promote interaction within an integrated, comprehensive repository network for aquatic model species repositories.
A microfluidic chip is described that facilitates research and quality control analysis of zebrafish sperm which, due to its miniscule (i.e., 2–5 μl) sample volume and short duration of motility (i.e., <1 min), present a challenge for traditional manual assessment methods. A micromixer molded in polydimethylsiloxane (PDMS) bonded to a glass substrate was used to activate sperm samples by mixing with water, initiated by the user depressing a transfer pipette connected to the chip. Sample flow in the microfluidic viewing chamber was able to be halted within 1 s, allowing for rapid analysis of the sample using established computer-assisted spermanalysis (CASA) methods. Zebrafish spermcell activation was consistent with manual hand mixing and yielded higher values of motility at earlier time points, as well as more subtle time-dependent trends in motility, than those processed by hand. Sperm activation curves, which indicate sample quality by evaluating percentage and duration of motility at various solution osmolalities, were generated with on-chip microfabricated gold floor electrodes interrogated by impedance spectroscopy. The magnitude of admittance was linearly proportional to osmolality and was not affected by the presence of sperm cells in the vicinity of the electrodes. This device represents a pivotal step in streamlining methods for consistent, rapid assessment of sperm quality for aquatic species. The capability to rapidly activate sperm and consistently measure motility with CASA using the microfluidic device described herein will help improve the reproducibility of studies on sperm and assist development of germplasm repositories.
Cryopreservation has been recognized as a powerful tool for long-term preservation of genetic resources. However, the outcomes of cryopreservation by different user groups often vary due to inconsistency in procedures and freezing equipment. Herein, we report on the feasibility of providing customizable sensing probes with three-dimensional (3-D) printing to monitor cryopreservation phenomena. The objectives were to: 1) introduce 3-D printing as a fabrication method for developing customizable probes to be used in cryogenic applications; 2) design and fabricate an example of a 3-D printed sensing probe and multiplexer capable of detecting phasechange phenomena based on quantitative data regarding sample electrical resistance and temperature, and 3) demonstrate the sensing platform in cryopreservation conditions and in combination with a custom-made 3-D printed freezing device. The sensing probe developed was designed to fit within standard 0.5-ml French straws. Phase-transition phenomena were detected by analyzing electrical resistance changes. The quantitative data from this device in conjugation with a 3-D printed freezer rack provided cryopreservation capability with high reproducibility and offered an alternative to expensive programmable freezers. The use of 3-D printing provided flexibility to develop new sensing probes or modify existing designs based on specific needs. After initial prototyping, fabrication, and testing of 3-D printed sensing probes, particularly useful designs can lead to the reduction of variation in performing standardized cryopreservation protocols.
Germplasm repositories are a necessary tool for comprehensive conservation programs to fully preserve valuable genetic resources of imperiled animals. Cryopreserved germplasm can be used in the future to produce live young for integration into other conservation projects, such as habitat restoration, captive breeding, and translocations; thus compensating for genetic losses or negative changes that would otherwise be permanent. Although hundreds of cryopreservation protocols for various aquatic species have been published, there are great difficulties in moving such research forward into applied conservation projects. Successful freezing of sperm in laboratories for research does not guarantee successful management and incorporation of genetic resources into conservation programs in reality. The goal of the present review is to provide insights and practical strategies to apply germplasm repositories as a real-world tool to assist conservation of imperiled aquatic species. Live-bearing (viviparous) fishes are used as models herein to help explain concepts because they are good examples for aquatic species in general, especially small-bodied fishes. Small live-bearing fishes are among the most at-risk fish groups in the world, and need urgent conservation attention. However, development of germplasm repositories for small live-bearing fishes is challenged by their unusual reproductive characteristics, such as formation of sperm bundles, initiation of spermatozoa motility in an isotonic environment, internal fertilization and gestation, and the bearing of live young. The development of germplasm repositories for goodeids and Xiphophorus species can provide examples for addressing these challenges. Germplasm repositories must contain multiple basic components, including frozen samples, genetic assessment and information systems. Standardization and process generalization are important strategies to help develop reliable and efficient repositories. An ideal conservation or recovery program for imperiled species should include a comprehensive approach, that combines major concerns such as habitat (by restoration projects), population propagation and maintenance (by captive breeding or translocation projects), and preservation of genetic diversity (by repository projects). In this context, strong collaboration among different sectors and people with different expertise is a key to the success of such comprehensive programs.
Vitrification is a method of cryopreservation that freezes samples rapidly, while forming an amorphous solid (‘‘glass’’), typically in small (lL) volumes. The goal of this project was to create, by three-dimensional (3D) printing, open vitrification devices based on an elliptical loop that could be efficiently used and stored. Vitrification efforts can benefit from the application of 3D printing, and to begin integration of this technology, we addressed four main variables: thermoplastic filament type, loop length, loop height, and method of loading. Our objectives were to: (1) design vitrification loops with varied dimensions; (2) print prototype loops for testing; (3) evaluate loading methods for the devices; and (4) classify vitrification responses to multiple device configurations. The various configurations were designed digitally using 3D CAD (Computer Aided Design) software, and prototype devices were produced with MakerBot 3D printers. The thermoplastic filaments used to produce devices were acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA). Vitrification devices were characterized by the film volumes formed with different methods of loading (pipetting or submersion). Frozen films were classified to determine vitrification quality: zero (opaque, or abundant crystalline ice formation); one (translucent, or partial vitrification), or two (transparent, or substantial vitrification, glass). A published vitrification solution was used to conduct experiments. Loading by pipetting formed frozen films more reliably than by submersion, but submersion yielded fewer filling problems and was more rapid. The loop designs that yielded the highest levels of vitrification enabled rapid transfer of heat, and most often were characterized as being longer and consisting of fewer layers (height). 3D printing can assist standardization of vitrification methods and research, yet can also provide the ability to quickly design and fabricate custom devices when needed.
The cryopreservation of genetic material can be an important tool for researchers and others involved with imperiled fishes, wild fisheries, aquaculture, and biomedical research. The standardization and reliable collection of diverse, high‐quality samples are persistent challenges to the successful cryopreservation of aquatic species. The overall goal of this study was to work with different user groups, cryopreserving sperm on‐site at their facilities to evaluate the uses and challenges of a mobile laboratory with high‐throughput and quality control capabilities comparable to those of a specialized central facility. The objectives were to demonstrate the collection and cryopreservation of sperm from (1) large‐bodied freshwater Blue Catfish Ictalurus furcatus for aquaculture; (2) small‐bodied freshwater swordtails and platyfishes Xiphophorus spp. for biomedical research and repository development for gametes from imperiled species; and (3) saltwater Red Snapper Lutjanus campechanus for wild fisheries research. Over the course of this project, the mobile laboratory traveled more than 4,000 km, collecting germplasm from more than 650 male fish. A total of 137 Blue Catfish were processed in 2015 and 2016, yielding 6,146 0.5‐mL French straws. A total of 521 males from 11 different species in the genus Xiphophorus were processed over 4 d in 2015, yielding 488 0.25‐mL French straws. Lastly, a total of 17 Red Snapper males were processed during 2015, yielding 316 0.5‐mL French straws. This study documents the development of a mobile cryopreservation laboratory with high‐throughput capability for aquatic species. If mobile laboratories prove to be effective, user groups will no longer be limited to germplasm resources that can be shipped as samples or transported as live animals to a central cryopreservation facility. Mobile laboratories would thus create opportunities to collect higher‐quality germplasm and provide access to new species. Also, they would enable direct cooperation, including training, among a wide variety of user groups for numerous applications.