Catfish Sperm Research Articles

Cryopreservation of Blue Catfish Spermatozoa and Subsequent Fertilization of Channel Catfish Eggs

Successful cryopreservation of fish spermatozoa has important implications for genomic conservation and for aquaculture. We report the first long-term cryopreservation of ictalurid sperm. Spermatozoa from nine blue catfish Ictalurus furcatus were cryopreserved for 12 months in three concentrations (3.75 × 108, 1.50 × 109, and 6.00 × 109 spermatozoa per straw), within two different straw sizes (0.5 and 1.0 mL), and with two cryoprotectants (DMSO or methanol combined with powdered skim milk). Cryopreserved spermatozoa were then used to fertilize lots of 450 eggs of channel catfish Ictalurus punctatus. Mean relative fertilization percentage for sperm treated with DMSO was 32% (range of 23–54%) of the fresh control. Spermatozoa frozen with the combination of an intracellular cryoprotectant (methanol) and an extracellular cryoprotectant (skim milk) produced no fertilization. No difference (P > 0.05) was observed between fertilization percentage of spermatozoa frozen in the two straw sizes. The highest level of relative fertilization percentage (54%) was achieved with the highest insemination dose, 6.00 × 109 spermatozoa per straw (P < 0.05). There was no difference (P > 0.05) in fertilization percentage between insemination doses of 3.75 × 108 and 1.50 × 109 spermatozoa per straw. Cryopreservation of blue catfish spermatozoa can be optimized by using the high concentration of sperm (6.00 × 109) and DMSO as cryoprotectant in either 0.5- or 1.0-mL straws.

The Effect of the Absence or Presence of Channel Catfish Males on Induced Ovulation of Channel Catfish Females for Artificial Fertilization with Blue Catfish Sperm

Abstract Female channel catfish Ictalurus punctatus were induced to spawn with carp pituitary extract either singly paired with channel catfish males in aquaria (males present) or stocked with multiple females in aquaria or tanks (males absent). There was no difference between ovulation rates of females induced to spawn in the presence or absence of channel catfish males (P = 0.57). Females hand-stripped with males absent produced more eggs per kilogram of body weight (4,585) than females with males present (2,942; P = 0.07). When females in the males-present treatment deposited eggs in the aquaria, which allowed observation of the readiness of females, the number of eggs available for stripping was reduced. There was no difference in fertilization rate between treatments when using sperm from blue catfish I. furcatus (P = 0.35). Females that ovulated with males absent produced a similar (P = 0.12) number of viable eggs per kilogram of female body weight as females that ovulated with males present (3,809 ...

A Preliminary Bacteriological Study of Refrigerated Channel Catfish Sperm

Abstract.— This study was designed to simulate conditions encountered routinely during refrigerated storage of channel catfish sperm. Sperm samples were stored at 4 C in non‐sterile and sterile Hanks' balanced salt 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 pseudomonad (Pseudomonas sp.) cultured from sperm samples stored in sterile buffer produced caseinase, lecithinase, and was β‐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.

Use of computer assisted sperm analysis (CASA) for monitoring the effects of pollution on sperm quality of fish; application to the effects of heavy metals

Computer assisted sperm analysis (CASA) has been used to analyse the effects of cadmium and zinc on the motility of fish sperm treated either for 24 h after partial dilution in an extender, or during the final dilution step on the microscope stage during video recording. The progressive motility of catfish sperm, which was not affected by storage for 24 h, was decreased after exposure to 100 ppm cadmium or 2000 ppm zinc in extender for 24 h. Only 1000 ppm cadmium or 2000 ppm zinc had any effect on motility when added to sperm at the final dilution stage. These results suggest that concentrations of cadmium which have been found to be present in fish gonads as a result of bioaccumulation from the water or diet may decrease the motility of sperm during maturation or storage in the testis. Cadmium toxicity was significantly decreased in the presence of an equal amount of zinc, suggesting an equilibrium between zinc and cadmium at binding sites in the sperm. Computer assisted sperm analysis is shown for the first time to provide a rapid and quantitative measure of the effects of environmental pollutants on the quality of fish sperm. It is suggested that the methodology described may form the basis for a standard toxicity test to assess the potential hazards of environmental pollutants on reproduction.

Spawning and management of gametes, fertilized eggs and embryos in Siluroidei

The structure of gonads and gametes is highly diversilied in siluroid fishes; in some groups the testes are composite with an anterior part formed of spermatogenetic tissues and the posterior part of seminal vesicles which may or may not store spermatozoa. For catfish species of aquacultural interest, ovaries present the same general morphology as in other teleosts with oviducts. Spermatozoa are biflagellated in the Channel catfish (Ictalurus punctutus) and monoflagellated in the European catfish (Silurus glanis) and African catfishes (Clarias gariepinus, Heterobranchus lingijilis). The egg size shows considerable intergroup differences, from 0.8-1.2 mm in some Pimelodidae and Clariidae up to 15-20 mm in Anidae. In numerous cases, the eggs are adhesive and develop either a surrounding sticky layer (Ictulurus, Chrysichthys, Silurus) or an adhesive attachment disc (Clarias, Heterobranchus). Fertilization is in general extemal but intemal fertilization is reported in some species. The annual sperm production was measured in European catfish and Channel catfish as 1.7 x 10 and 1.8 x 10 spermatozoa.kg-' body weight, respectively. In females, the fecundity ranges from about 50 eggs per spawn in ariids up to more than 100000 eggs.kg-' body weight, e.g. in pimelodids and clariids. A large variety of controlled reproduction systems are found in the siluroids. In some cases (Ictulurus, Chrysichthys, Hoplosternuin), spawning oceurs naturally in ponds or tanks provided that adequate spawning substrates are available; fertilized eggs are collected immediately after spawning and placed into incubators until hatching. But for most species, although natural or seminatural spawning could be achieved in captivity, the tendency at the present time is to develop techniques using hormonal-induced ovulation and artificial insemination in order to control the various steps of reproduction and to allow gamete preservation and manipulation. Oocyte maturation and ovulation or spermiation can be induced in many species by a large variety of hormones (GnRHs, fish gonadotropins, hCG, vanous steroids etc). Some information is available on gamete biology and preservation. Sperm motility is short-lived, not exceeding 70-80 s of fonvard movement as in most other freshwater teleosts. A peculiarity of the European catfish sperm is the activation by urine during sampling which could be prevented by direct collection in an immobilizing solution (NaCI 200 mM, Tris HCl 30 mM, pH 7.0). Spermatozoa cryopreservation was successfully attempted in several species. Ova generally survive only a few hours after ovulation and fertilization must be camed out soon after. Methods for gamete collection, insemination and incubation of eggs are descnbed for the most widely cultured siluroid species.

Refrigerated Storage of Channel Catfish Sperm

Abstract.— 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 Icralurus 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: 1) 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‐loc® plastic bags without supplemental oxygen (7 d), or sperm stored in plastic beakers (8 d) or test tubes (8 d) without supplemental oxygen. Sperm stored with the addition of antibiotic/antimycotic cocktail or methanol retained motility significantly longer (10–12 d) than did sperm stored without additives (6–8 d). Sperm stored in extender solution without glucose retained motility significantly longer (19–21 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.

Effect of Osmotic Pressure on the Activation and Storage of Channel Catfish Sperm

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 (1) 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) was estimated in diluted Hanksˈ 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 about 10%. 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 plays a major role in the activation of channel catfish sperm. Sperm stored in 122 mosmol/kg HBSS lost 82% 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.

Effects of sperm concentration and egg number on fertilization efficiency with channel catfish ( [formula omitted]) eggs and blue catfish ( [formula omitted]) spermatozoa

The mean sperm concentration of 10 blue catfish ( Ictalurus furcatus ) was 1.03 × 10 10 per gram of testis. Testis weighed 3.9 and 17.2 g, with a mean of 6.6 g per fish. Fertilization rate of channel catfish ( Ictalurus punctatus ) eggs fertilized with 5.00 × 10 4 to 1.20 × 10 7 blue catfish spermatozoa per egg was 17 to 87%, with an overall mean of 65%. Sperm concentrations of 5.0 × 10 4/egg exhibited a lower, 16.6% (P < 0.05) fertilization rate than higher sperm concentrations (1.25 × 10 5 to 1.20 × 10 8/egg). Batches of 450, 2,000, 5,000, 8,000 and 11,000 eggs were similarly fertilized with various sperm concentrations. Mean fertility rate ranged from 25 to 67%, with an overall mean of 53%. The largest egg mass produced the lowest (P < 0.05) fertilization rate. A combination of 450 eggs per batch and 5.0 × 10 5 to 1.20x 8 sperm per egg produced the highest rate of fertilization (67 to 87%).

Standardization of Ultraviolet Irradiation of Channel Catfish Sperm

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 gynogenesis has been used in the breeding and genetic study of several fishes, including salmonids (Chourrout 1982; Allendorf et al. 1986), tilapia (Don and Avtalion 1988), and channel catfish (Liu et al. 1992). Gynogenesis has been applied to production of monosex 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.

Cryopreservation of Channel Catfish Sperm: Storage in Cryoprotectants, Fertilization Trials, and Growth of Channel Catfish Produced with Cryopreserved Sperm

Abstract 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 –196°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 ...

The Effect of Freezing Rate on the Survival of Cryopreserved African Sharptooth Catfish ( Clarias gariepinus) Spermatozoa

Cryoprotective agents were evaluated to find the optimal concentration of the cryoprotectant and most suitable combination of solution and cryoprotectant. A cryoprotective agent composed of 4% glucose and 9% glycerol yielded the best results. It was established that the optimal freezing rate is dependent on the composition of the cryoprotective agent. Maximal survival of catfish spermatozoa (60%) occurs at 5°C min -1 and faster and slower freezing rates result in poor survival or no survival at all. Incorporation of an isothermal holding period into the freezing rate led to remarkable increase (20-30%) in sperm survival when Me 2SO was present in the cryoprotective agent. Cryoprotective agents containing glucose also showed improved survival when a three phase freezing rate was used. These results lead to the conclusion that the presence of an isothermal holding period in the freezing rate is beneficial for the cryoprotective action of Me 2SO and glucose.

Preservation of Channel Catfish Sperm

Abstract Three activators, eight extenders, and three protective agents at two concentration levels each were used to determine the best combination that will maintain in storage the viability of channel catfish (Ictalurus punctatus) sperm cells. There was a highly significant (P < 0.01) difference in motility score among activators. Saline (0.65%) gave the highest motility score. Extended sperm cells became motile when activated after 2 months of storage at 4 C. Extender I (Truscott and Idlers Hfx #1) with 10% dimethyl sulfoxide (DMSO) and 2 hours equilibration produced the highest motility scores after freezing of 24 hours or 1 week. Freezing rate was controlled by placing vials of sperm in a double-layered, kapok-lined paper bag and freezing at the height of 127 mm above the liquid nitrogen level.

Spermatozoal Morphology and Ultrastructure of Channel Catfish, Ictalurus punctatus

Abstract The morphology, dimensions, aberrations, and ultrastructure of channel catfish (Ictalurus punctatus) spermatozoa were studied. Spermatozoa of a wild catfish stock from Louisiana were compared to those of a domestic stock from Mississippi. The spermatozoon of channel catfish consists of a rounded dark head, a collar-dike midpiece, and a long flagellum. The average dimensions were: head length, 2.3 μm; midpiece length, 1.6 μm; midpiece width, 3.1 μm; and flagellum length, 94.9 μm. The flagellum had the classical (2.9 + 2) axoneme. On the average, 4.7% of the spermatozoa in the fish studied were biflagellar. Each flagellum arose from an individual centriolar complex. Domestic fish stock had a higher percentage of normal gametes compared to wild stock.