Cryopreservation of YamúBrycon siebenthalae Milt

The yamú Brycon siebenthalae is an endemic fish of the Orinoco river basin, but wild stocks are decreasing because of the disruption of their habitat. We evaluated a protocol for the cryopreservation of yamú sperm to contribute to the preservation of this endangered genetic resource. Milt was mixed with a cryoprotectant medium (5.5% glucose, 12% egg yolk, and 5%, 10%, or 15% dimethyl sulfoxide - DMSO) in a ratio 1:4 (milt:medium), stored in 0.5-mL French straws, frozen in nitrogen liquid vapor (-76 C), then immersed and stored in liquid nitrogen for 10 d or 12 mo. Motility of thawed spermatozoa was higher ( P < 0.001) in 10% DMSO medium than 5% DMSO or 15% DMSO mediums; but lower than the control ( P < 0.001). With sperm cryopreserved, the highest level of fertilization was achieved with 10% DMSO ( P < 0.001) after 10 d or 12 mo of cryopreservation. Fertilization of eggs inseminated with 6.4 × 10⁹ spermatozoa per g of eggs was higher ( P <0.05) than with 1.6 × 10⁹ spermatozoa per g of eggs. There was no difference (P > 0.05) in fertilization between insemination doses of 3.2 × 10⁹ and 6.4 × 10⁹ spermatozoa per g of eggs. Cryopreservation of yamu milt can be performed successfully with a simple medium combined with 10% of DMSO as cryoprotectant. The highest level of fertility was achieved using between 3 × 10⁹ and 6 × 10⁹ spermatozoa per g of fresh eggs.     

Artificial insemination in tench, Tinca tinca L.

Abstract. The sperm of tench, Tinca tinca L., is characterized by a milky colour and consistency, and is of very low density. After collecting the sperm, motion of spermatozoa was recorded even without water activation. A better motility value (value 4·36 on average) was observed in spermatozoa collected in immobilizing solution (collecting medium) and stored for 3h, when compared with spermatozoa without collecting medium. Average total and relative numbers of spermatozoa were 12·16 × 10⁹ per male and 18·50 × 10⁹ per kg of body weight, respectively. When testing the effect of activating solution in artificial propagation of tench, the highest fertilization rates (81·3 and 85% in two cases) were found for NaCl solution with an osmotic concentration of 34 or 69 mOsmol and for fresh water, respectively. The fertility rate was reduced significantly ( P < 0·01) by any increase above 105 mOsmol in NaCl concentration in the activating solution. In the tests of optimal method of artificial fertilization, the highest hatching rate of sac fry (71·35%) was found in sperm collected into immobilizing solution. The application of immobilizing solution significantly increased the number of sac fry at the levels P < 0·1 and P < 0·01, if compared with intact sperm stored for 3 h and fresh sperm, respectively.     

Vaccination of Mixed and Full-Sib Families of Channel Catfish Ictalurus punctatus Against Enteric Septicemia of Catfish With a Live Attenuated Edwardsiella ictaluri Isolate (RE-33)

These studies were conducted to evaluate the efficacy of a live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish. In one study channel catfish fingerlings (72 d of age post hatch) were immersed for 30 min in water containing E. ictaluri RE-33 at dosages of 1 × 10⁶, 1 × 10⁷ and 2 × 10⁷ CFU/ML of water. No mortalities were observed following vaccination. Following exposure to virulent Edwardsiella ictaluri the cumulative mortality of fish vaccinated with dosages of at least 1 × 10⁷ CFU/mL were significantly lower than that of non-vacccinated fish in both laboratory and field challenges. Vaccination with 1 × 10⁶ CFU RE-33mL provided some protection during the laboratory challenge but failed to protect fish under field conditions. In a second study, vaccination of 6 full-sib families of channel catfish at a vaccine dosage of 1 × 10⁷ CFU/mL resulted in a relative percent survival among families ranging from 67.1 to 100%. Significant differences in mortality were found among families and between vaccinated and unvaccinated groups, but there was no family by vaccine interaction. Families with the highest mortality after vaccination were also shown to have the highest mortality without vaccination (r = 0.82; P = 0.04).     

Effects of high dose of vitamin A on reproduction and egg quality of Japanese flounder Paralichthys olivaceus

ABSTRACT: The present study was conducted to investigate the effects of dietary vitamin A on reproduction and egg quality in Japanese flounder Paralichthys olivaceus . Broodstock were fed experimental pellets containing two levels of vitamin A [11 × 10³ IU/100 g (control diet; CD), 337 × 10³ IU/100 g (experimental diet; ED)] for approximately 2 months before spawning and during the spawning period. Two groups of five females (average weight 1.4 kg) and 10 males (average weight 0.7 kg) were randomly allocated to two 30 m³ indoor tanks. Total egg production of the CD group was slightly higher than the ED group. Percentage of buoyant eggs and hatching rate of the ED group were significantly higher than the CD. In other egg quality parameters, such as percentage abnormal larvae and starvation tolerance of larvae, no notable difference was found between these two groups. At the end of the experiment, the skin color of broodstock in the ED group was darker than that of the CD group. Vitamin A content in eggs of the ED group was significantly higher than that of the CD group. However, the difference in vitamin A content in eggs between the ED and CD groups was much smaller than that in the liver of the females between the two groups. These results indicate that feeding broodstock a higher level of vitamin A increases the vitamin A content in eggs but does not affect egg quality in Japanese flounder because excess dietary vitamin A was stored mainly in the broodstocks' liver.     

Influence of photoperiod, frequency of stripping and presence of females on sperm output in turbot, Scophthalmus maximus (L.)

Abstract. The effect of four environmental conditions was investigated upon sperm output in turbot, Scophthalmus maximus (L.), submitted to three different rhythms of stripping. Males kept under a natural light cycle and under a 6-month contracted light programme released a similar sperm output in terms of total volume of semen produced per fish during the experimental period (4·9 ± 0·9ml), mean sperm concentration (29·4 ± 2·8 × 10⁹ spermatozoa/ml) and total sperm number (163·2 ± 40·5 × 10⁹ spermatozoa). Attempts to stimulate spermiation for a second time just after the end of the natural reproduction period resulted in the release of low sperm output (total volume of semen: 1·6 ± 0·4 ml; mean sperm motility: 2 min 36s ± 0 min 47s; mean sperm concentration: 47·6 ± 10·2 × 10⁹ spermatozoa/ml; total sperm number: 84·5 ± 25·3 × 10⁹ spermatozoa). Stripping frequency had no effect on total volume of semen, mean sperm motility and total sperm number. Monthly collection did not modify sperm samples in relation to stripping rank. However, decreasing volume, motility and sperm concentration were observed when males were stripped fortnightly and weekly. During the natural spawning period, the presence of females in the tank enhanced mean sperm motility (from 3 min 27s + 0 min 52s to 6 min 38s ± 1 min 38s).     

Temporal variation of seasonality of egg production and the spawning biomass of Pacific anchovy, Engraulis japonicus, in the southern waters of Korea in 1983–1994

Embryonic mortality, egg production and the spawning stock biomass of Pacific anchovy, Engraulis japonicus , off Southern Korea during 1983–1994, and their biological response to oceanographic features in spring and summer, were analysed. The instantaneous mortality rate (IMR) of embryonic stages decreased in spring and increased in summer, with a range of 0.33–1.23 day^–1 in spring and 0.78–1.69 day^–1 in summer. Egg production in summer was three times that during spring and production was low in the late 1980s. Mean lengths of yolk-sac larvae and adult females were greater in spring than in summer, whereas spawning fraction and spawning stock ratio (spawning biomass:adult biomass) were lower in spring than summer. Estimated mean spawning stock biomass ranged from 141 × 10³ to 380 × 10³ MT in spring and from 221 × 10³ to 557 × 10³ MT in summer. Statistically, the seasonal and long-term trends of embryonic mortality, egg production and spawning stock biomass of Pacific anchovy can be explained largely by spring warming, summer cooling and by less abundant zooplankton in the late 1980s.     

Cryopreservation of Sperm of Farmed European Eel Anguilla anguilla

Sexual maturation and sperm release were induced in farmed European eels Anguilla anguilla kept exclusively in fresh water by using two dosages of human chorion gonadotropin (100 International Unit (IU)-Group one and 250 IU/individual per week-Group two). Sperm release took over 13 wk in both groups. The quality of sperm was investigated on the eighth, ninth, and tenth wk. The average cell densities were 0.94 ± 0.4 × 1010 (Group one) and 0.93 to. ± 0.6 × 10¹⁰ (Group two) spermatozoa/mL. The estimated motility of eel sperm was 33, 55, and 49% on the eighth, ninth, and tenth wk of treatment, respectively. The estimated average motility of samples selected for cryopreservation was 73 ± 10%, while the post-thaw motility of cryopreserved samples was 36 ± 11%. The extender originally developed for common carp sperm crypreservation together with methanol as cryoprotectant was found suitable for the cryopreservation of European eel sperm.     

Maturation and artificial fertilization of eggs of captive common wolf fish, Anarhichas lupus L. from the White Sea

Abstract. A stock of mature common wolffish, Anarhichas lupus L., were kept in tanks at the surface temperature in the sea and at a lowered temperature. At temperatures of 5–8C in August and September all females matured. The fertilization rate of the eggs was studied in relation to various aspects of the fertilization procedure including sperm concentration, the duration of the contact of eggs with the sperm and the time of exposure of the gametes outside of the body of spawners. The maximum fertilization rate was attained at sperm concentrations not less than 1.0 times 10⁶ per ml and when eggs were exposed to sperm for 1–2h. Fertilization of eggs in a narrow cylindrical vessel improved the efficiency of fertilization, with rates of up to 80–95%. Features of the fertilization of eggs of the wolffish are discussed in relation to the reproductive biology of the species.     

Age-Related Sperm Quality of Captive Striped Bass Morone saxatilis

Three groups of captive-reared striped bass Morone saxatilis ages 1, 3 and 12 yr, were examined for age-related changes of sperm characteristics including short-term storage. All groups had similar ranges of the following parameters (mean× SEM): expressible milt (5.6× 0.5 mI/kg body weight (BW) to 7.5× 2.1 mL/kg BW), percentage of motile sperm (55× 6% to 60× 2%), duration of sperm motility (69× 3 sec to 72× 5 sec) and percentage of viable sperm (91× 2% to 93× 2%). Compared to the 1 and 12-yr-old fish, the 3-yr-old fish produced the greatest number of spermatozoa (1,190× 370× 10⁹ spermatozoa/kg), sperm concentration (120× 8 × 10⁹ spermatozoa/mL) and spermatocrit (74× 4%). In addition, during short-term storage at 4 C, extender-preserved sperm samples of the 3-yr-old group showed a significantly higher ( P < 0.05) percentage of motile sperm and duration of sperm motility, compared to the other two groups. This suggests that short-term storage may be affected by the age of the male fish. Sperm longevity of the 3-yr-old group was successfully maintained for as long as 15 d, longer than that of the 1-yr-old group (9 d) and 12-yr-old group (7 d). Overall, the 3-yr-old fish appeared to have superior sperm quality than the 1 or 12-yr-old fish based on higher sperm production and increased sperm longevity.     

Effects of dry pellets containing astaxanthin and squid meal on the spawning performance of striped jack Pseudocaranx dentex

ABSTRACT: This study was conducted to investigate the effect of squid meal and astaxanthin in dry pellets on the spawning performance of striped jack. Two groups of 8-year-old fish with an average bodyweight of 3.5 ± 0.4 kg were used. The control group was fed a raw fish mix (RF) and the experimental group was fed steam dry pellets (as-DP) containing 10 p.p.m. astaxanthin and squid meal, the latter replacing half the fish meal. Each group was fed once a day, three times a week in floating net cages for a period of 5 months. Seven females and seven males were selected from each group and transferred to 65 m³ indoor concrete tanks for spawning, where they were kept at optimum spawning conditions (22°C) for 1 month. The average bodyweights of selected fish were 3.8 ± 0.5 kg and 3.8 ± 0.6 kg for the RF and as-DP groups, respectively. Both groups spawned a total of 18 times and the mean total egg production and egg quality, measured in terms of buoyancy, fertilization and hatching rates, showed no significant differences ( P > 0.05) between groups. Total larval production was 679.9 × 10³ and 678.1 × 10³ larvae/kg-female for the RF and as-DP groups, respectively. These results show that the combination of astaxanthin and squid meal in dry pellets for striped jack broodstock improved the overall spawning performance to a level similar to that of fish fed on RF.     

Preliminary investigations on the effects of dietary lipid on the spawning performance and egg quality of black sea bass Centropristis striata L

Adult black sea bass Centropristis striata broodstock ( N =162) were fed three different dietary treatments: two commercially prepared diets with 45% protein and two different lipid levels (12% and 20%) (diets 1 and 2), and a diet of frozen Atlantic silversides Menidia menidia (SS, diet 3). Broodstock were held under controlled photothermal conditions and induced to spawn with an LHRHa pellet (72 μg kg⁻¹ bw). Dietary lipid had pronounced effects on spawning performance and egg quality. Diet 3 (SS) produced a significantly ( P <0.05) higher fertilization success (22.4%) than diets 1 (0.6%) and 2 (4.8%). The hatching success of fertilized eggs was similar in all diets (range=40–58.6%), but only two spawns from diet 1 (12% lipid) yielded viable yolk-sac larvae (YSL). Diet 3 (SS) also produced significantly more YSL per female (21.8 × 10³) than the diet 1 (0.3 × 10³). Eggs from diet 3 (SS) contained a significantly greater proportion of n-3 series fatty acids, with docosahexaenoic acid (DHA) as the largest fraction. Eggs from commercially prepared dietary treatments contained significantly more n-6 fatty acids. The poor spawning performance of fish fed diet 1 (12% lipid) may be related to higher levels of linoleic acid and lower levels of DHA in the diet.     

The susceptibility of salmonid fish to an atypical strain of Aeromonas salmonicida that infects goldfish, Carassius auratus (L.), in Australia

Abstract. An enzootic, Australian, atypical strain of Aeromonas salmonicida isolated from diseased goldfish, Carassius auratus (L.), was inoculated into Atlantic salmon, Salmo salar L., brown trout, S. trutta L., rainbow trout, S. gairdneri Richardson, and brook trout, Salvelinus fontinalis (Mitchill), fingerlings by intraperitoneal injection (i.p.) and by bath challenge, the latter with and without prior abrasion of skin. The 10-day LD₅₀ (i.p.) was estimated to be 7·4 × 10^-3 colony forming units (cfu) for Atlantic salmon, 3·0 × 10^-2 cfu for brown trout, 3·7 × 10² cfu for brook trout and 6·4 × 10³ cfu for rainbow trout. Brown, rainbow and brook trout succumbed to bath challenges with between 10⁵–10⁶ cfu/ml, developing ulcers of the skin and septicaemia. The organism was trasmitted from inoculated fish to five of 195 within-tank control fish via water and established a carrier state in one of 14 Atlantic salmon. It was concluded that the organism poses a significant threat to the salmonid farming industry and wild salmonid fisheries in Australia.     

Comparative sensitivity of carp, Cyprinus carpio L. and rainbow trout, Salmo gairdneri Richardson, to Renibacterium salmoninarum

Abstract. The pathogenicity of Renibacterium salmoninarum to carp, Cyprinus carpio L., and rainbow trout, Salmo gairdneri Richardson, was investigated. All carp injected with 4·8 × 10⁸ cells/fish, or 4·8 × 10⁷ cells/fish survived for 38 days. R. salmoninarum was isolated from all moribund fish, but not from the kidney of surviving fish, although R. salmoninarum antigen was detected in several of these fish by the dot blot assay. On the other hand, mortality in rainbow trout was 95% in the fish injected with 4·8 × 10⁸ cells/fish, and 15% in those which received 4·8 × 10⁷ cells/fish. R. salmoninarum antigen was detected by the dot blot assay in all surviving rainbow trout. The number of R. salmoninarum cells was immediately decreased by carp or rainbow trout serum, and the serum bactericidal activity of carp was higher than that of rainbow trout. Carp blood leucocytes had higher phagocytic activity than those of rainbow trout.     

Natural Spawning of Captive Bigeye Scad Selar crumenophthalmus in Hawaii

Abstract.— Juvenile bigeye scad Selar crumenophthalmus were caught from the wild by hook and line and reared over 3 yr to establish a spawning broodstock of this species. The size at first maturity for captive males and females was 19-cm and 25-cm fork length (FL). Natural spawning of captive individuals occurred during the first year in captivity. The broodstock were observed to spawn repeatedly throughout their second and third years in culture. This species is a multiple spawner producing approximately 96 × 10³ to 121 × 10³ eggs/spawning group, 5 to 10 times per year. Stocking densities of 1–8 individuals/m³ were found to be suitable for natural spawning to occur. Collectively, the results indicate that this species exhibits notable potential for artificial propagation.     

Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10¹, 10³ or 10⁵ plaque forming units (pfu) L⁻¹ of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L⁻¹ (low density) or 140 fish L⁻¹ (high density), and the tank flow rate was 250 mL⁻¹ min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L⁻¹ exposure did not occur until IPNV concentration was 10⁵ pfu L⁻¹ at low fish density and 10³ pfu IPNV L⁻¹ at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are ≤10³ pfu IPNV L⁻¹. In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10¹ pfu L⁻¹ may result in IPN epidemics in aquaculture facilities.     

An experimental investigation on aspects of infectious salmon anaemia virus (ISAV) infection dynamics in seawater Atlantic salmon, Salmo salar L.

This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 × 10¹ TCID₅₀ mL⁻¹ indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 × 10⁻² TCID₅₀ mL⁻¹ kg⁻¹) and rose to levels above the minimum infective dose (4.2 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹ 15 days post-infection. These data provide important information relevant to the management of ISA.     

The infectivity by different routes of exposure and shedding rates of Aeromonas salmonicida subsp. salmonicida in Atlantic salmon, Salmo salar L., held in sea water

Abstract. The infectivity of the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida to Atlantic salmon, Salmo salar L., in sea water was investigated and found to be similar to that reported for fresh water. The minimal infective dose in short duration bath exposures (1–3 days) was 10⁴ colony-forming units (cfu) per ml, while prolonged exposure for three weeks, but not for 1 week, produced infection with 10² cfu/ml. Intragastric intubation of A. salmonicida established infection with doses of >10⁵ cfu. Release of bacteria from dead or morbid infected fish was monitored and found to be in the order of 10⁵–10⁸ cfu/fish/h. These results emphasize the importance of removing dead fish from farm sites.     

Effects of temperature, salinity and irradiance on the growth of the toxic dinoflagellate Gymnodinium catenatum (Dinophyceae) isolated from Hiroshima Bay, Japan

ABSTRACT: Temperature and salinity ranges in which Gymnodinium catenatum (Hiroshima Bay strain) showed specific growth rates higher than 0.2/day were approximately 20–30°C and 20–32. The specific growth rate (μ), expressed as a polynomial equation as functions of temperature ( T ; °C) and salinity ( S ) were μ = (−6.84 × 10⁻⁴ T ² + 0.0354 T – 0.213) × (−1.03 × 10⁻³ S ² + 0.0579 S – 0.548)/0.31; the maximum growth rate (0.31/day) was obtained at 25°C and 30. From a comparison with field data recording temperature, salinity and light intensity, this species may be expected to bloom from summer to autumn in Hiroshima Bay.     

Evaluation of Sperm-Activating Solutions in Atlantic Salmon Salmo salar Fertilization Tests

The use of saline solutions was tested to determine their efficacy as replacements for ovarian fluid as sperm activators and to eliminate variability encountered with the use of Ovarian fluid. We tested fertilization rate of semen from eight males on Atlantic salmon Salmo salar eggs after five sperm-activating solutions and a non-activating saline were substituted for ovarian fluid. We used solutions shown acceptable for use with other salmonid species. The six solutions tested were a non sperm-activating phosphate-buffered saline (PBS, 7.2 g/L NaCl, 1.48 g/L Na₂HPO₄, 0.43 g/L K H₂PO₄), a Tris buffer (6.99 g/L NaCl, 3.63 g/L Tris and 2.42 g/L glycine), a Borax buffer (12.2 g boric acid/L in solution 1, 76 g disodium tetraborate/L combined 100:118, then 1 L combined with 3.7 L water and 18 g NaCl), and three solutions of 9.25 g/L (125 mM) NaCl buffered to pH 6.0, 7.5, and 8.9. The latter five solutions activated sperm immediately on contact, while PBS required additional water to activate sperm. The PBS solution was the least effective (mean percent eyed eggs 37.6%) for egg fertilization. The mean percent eyed eggs for the other five saline solutions (range 78% to 86%) were not significantly different. Sperm from one male provided significantly lower egg fertilization (39.6%) when compared with the other seven males (67.2–87.4% egg fertilization). Percent egg fertilization was not related to number of live sperm cells per egg. Our results show that osmotically-balanced sperm-activation solutions, even those with a pH range from 6.0 to 8.9 provide adequate media for fertilization of Atlantic salmon eggs. Fertilization in a deactivation saline and water reactivation of sperm resulted in low egg fertilization.