Short-term storage and cryopreservation of milt from Atlantic salmon and sea trout

Experiments on short-term preservation of sperm were performed with Atlantic salmon (Salmo salar). Fertility was maintained for up to 10 days when 2 mm thick samples were stored at 0° C under an oxygen atmosphere in the presence of antibiotics (125 IU penicillin and 125 μg streptomycin per ml sperm). Fertility was completely lost after 24 days. Sperm stored without antibiotics fertilized 100% of eggs after 6 days.

Cryopreservation was carried out with milt from Atlantic salmon and sea trout (Salmo trutta). Semen mixed with extender was frozen on dry ice (pellets) with subsequent storage in liquid nitrogen. Sperm pellets were thawed in a 0.12-M NaHCO₃-solution (10° C) before insemination. The suitability of an extender as previously described by Stoss and Holtz and of a 0.3-M glucose solution with the addition of 10% DMSO, was tested on two different batches of sperm and eggs in Atlantic salmon and sea trout. In addition, the extender earlier reported by Mounib and an aqueous solution of 10% DMSO were only used in Atlantic salmon with one batch of gametes.

Insemination with cryopreserved Atlantic salmon sperm resulted in 36 to 91.3% eyed eggs (control = 100). The differences were caused by the type of extender and the batch of gametes employed. The very simple extender consisting of 0.3 M glucose and 10% DMSO only was the most successful. Results with cryopreserved sea trout sperm ranged between 38.6–54.8% eyed eggs, showing no difference between treatments.     

Fertilization efficiency of cryopreserved sperm from striped catfish, Pangasius hypophthalmus (Sauvage)

The fertilization efficiency of cryopreserved sperm was compared with fresh sperm from striped catfish, Pangasius hypophthalmus . Of the two sets of experiments carried out, the first compared four sperm doses using fresh sperm and fresh eggs. The second experiment compared six concentrations of cryopreserved sperm ranging from 6.94 × 10⁷ to 6.94 × 10¹⁰ to fertilize 100 eggs per batch. Fertilization, hatch and survival rates were compared between cryopreserved and fresh sperm. The highest fertilization rate (53.75±1.62%) was achieved with a sperm dose of 6.94 × 10⁸. Increasing the sperm dose to 3.47 × 10⁹ did not increase the fertilization rate, indicating that the optimum sperm:egg ratio lies between 6.94 × 10⁶ and 3.47 × 10⁷ sperm per egg. Both highest (6.94 × 10¹⁰) and the lowest (6.94 × 10⁷) sperm doses resulted in lower fertilization rates (2.04% and 16.90% respectively). No significant differences were found among four fresh sperm doses compared. Mean hatch and survival rates resulting from fresh and cryopreserved sperm were similar. The experiment shows that while only 1.89 × 10⁶ fresh spermatozoa was required to fertilize a fresh egg, 6.94 × 10⁶ (or 3.67 times more) cryopreserved sperm was required to achieve the same level of fertilization. This provides important information for making decision to cryopreserve sperm for commercial and/or conservation purposes.     

Preventive efficacy of sodium hypochlorite against water mold infection on eggs of chum salmon Oncorhynchus keta

ABSTRACT:   To protect chum salmon eggs from water mold infection during incubation, the eggs were treated daily with sodium hypochlorite solution (NaOCl) at 10 mg/L residual chlorine concentration for 15 min during their developmental period from fertilization to eyed stages. The number of infected eggs and number of eyed eggs were observed on day 23 of incubation. The percentage of infected eggs to total eggs was significantly lower with NaOCl treatment (1.8 : 33.4%) than in the control (11.3 : 59.3%, P  < 0.01). The percentage of eyed eggs to total eggs was significantly higher with NaOCl (85.9 : 98.6%) than in the control (66.1 : 97.5%, P  < 0.01). The antifungal activity of NaOCl resulted in improving egg survival. Accordingly, NaOCl is a useful antifungal agent against water mold infection on chum salmon eggs.     

An efficient method for cryopreservation of testicular sperm from the Northern pike, Esox lucius L.

The cryopreservation of semen from the Northern pike, Esox lucius L., was investigated with a method that was originally developed for the Salmonidae. Because the amounts of semen obtained by stripping were insufficient, the suitability of testicular sperm was tested for cryopreservation. Frozen-thawed testicular sperm had fertilization rates similar to frozen-thawed semen obtained by stripping (74.2-84.7%), and at sperm to egg ratios of S= 4.5 × 10⁵ spermatozoa per egg, the post-thaw fertilization rates were also similar to fresh, untreated semen controls. Out of all the fertilization solutions investigated, a 100-mm NaCl, 10-mm Tris (pH 9) solution resulted in the highest post-thaw fertilization rates. To facilitate the fertilization of large egg batches, 1.2-mL straws were used for cryopreservation with a similar efficiency to 0.5-mL straws.     

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.     

Application of Computer‐assisted Sperm Analysis in Selecting the Suitable Solution for Common Carp,Cyprinus carpio L., Sperm Motility

The common carp, Cyprinus carpio L., sperm motility parameters were analyzed by using computer‐assisted sperm analysis system. The percentage of motile sperm (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity (VCL, µm/sec), average path velocity (VAP, µm/sec), the wobbling index (WOB, %), movement linearity (LIN, %), beat cross frequency (BCF, Hz), and amplitude of lateral head displacement (ALH, µm) were determined. Five activation solutions (As) were used to activate sperm movement. As 1 solution: 68 mM NaCl, 50 mM urea, 0.5% bovine serum albumin (BSA), pH: 7.7, 181 mOsm/kg; As 2 buffer: 100 mM NaCl, 10 mM Tris, 0.5% BSA, pH: 9.0, 199 mOsm/kg; As 3 solution: 86 mM NaCl, 0.5% BSA, pH: 7.4, 167 mOsm/kg; As 4 buffer: 5 mM KCl, 45 mM NaCl, 30 mM Tris, 0.5%, pH: 8.0, 160 mOsm/kg; and As 5 solution: distilled water with the addition of 0.5% BSA, pH: 7.3, <3 mOsm/kg. Among five tested solutions, a buffer with a pH of 9.0 and osmolality of approximately 200 mOsm/kg (As 2) was the most suitable. After its activation, a significant increase in MOT and ALH values was observed, which can be of importance to the effectiveness of egg fertilization .     

Dietary phosphorus requirement of juvenile Atlantic salmon, Salmo salar L.

The objective of this study was to determine the dietary phosphorus (P) requirement of juvenile Atlantic salmon, Salmon salar L. Triplicate groups of fish (mean initial weight 1.4 g) were fed semipurified, casein-gelatine-based diets containing one of five levels of P (4, 8, 10, 15 and 25 g kg⁻¹) from Ca(H₂PO₄)₂·H₂O, or a commercial feed (17 g kg⁻¹ P) for 9 weeks. Weight gains did not differ significantly among treatment groups fed the experimental diets but were slightly less than gains in fish fed the commercial feed. Feed efficiency (wet weight gain/dry feed consumed) was similar in all groups, averaging 1.45. Availability of dietary P, estimated from apparent retention and apparent digestibility, was 86%. Whole-body P concentrations declined in fish fed diets containing less than 10 g kg⁻¹ P. Fitting a logistic curve to dietary P vs. whole-body P concentrations indicated that a minimum of 11 g kg⁻¹ dietary P (9 g kg⁻¹ digestible P) was required by juvenile Atlantic salmon to maintain whole-body P concentrations at initial levels. Calculation of a dietary requirement using a simple factorial model which incorporated measurements of P availability, feed efficiency and normal whole-body P concentration indicated that the dietary requirement was approximately 10 g kg⁻¹. The dietary requirement established in this study (10–11 g kg⁻¹) is higher than previously reported for Atlantic salmon or other fishes. Possible reasons for the wide range of reported dietary P requirements in fishes are discussed.     

哲罗鲑卵子和卵腔液的生化组成与其发眼率的相关性

为了解哲罗鲑(Hucho taimen)卵子及卵腔液的生化组成及其与发眼率的关系,采用生物化学的方法对雌性哲罗鲑Ⅴ期卵子及卵腔液的酶、蛋白质﹑氨基酸﹑维生素和矿物质等组成和含量进行了测定,并且将各组份与哲罗鲑受精卵的发眼率进行了回归分析。结果表明:哲罗鲑Ⅴ期卵子中含有碱性磷酸酶、酸性磷酸酶、谷草转氨酶、琥珀酸脱氢酶﹑Mg2+-ATP酶﹑磷﹑钙离子﹑铁离子﹑维生素C和维生素E。卵子中Mg2+-ATP酶活力≥8.3×10-5μmol(Pi)/(min.mg prot)、磷含量在12.97~23.0 3μmol/g(卵)之间及氨基酸含量在574.89~1195.40μmol/g(卵)之间,哲罗鲑卵的发眼率均≥80%。哲罗鲑的卵腔液中含有碱性磷酸酶、酸性磷酸酶、谷草转氨酶、琥珀酸脱氢酶、磷、钙离子、铁离子、维生素C和维生素E,当谷草转氨酶酶活力≥18.14μmol/(min·L),哲罗鲑卵的发眼率≥80%。哲罗鲑Ⅴ期卵子中Mg2+-ATP酶活力、磷含量﹑氨基酸含量以及卵腔液中谷草转氨酶酶活力与其发眼率存在着显著相关性。     

Reproductive biology,early ontogeny,and effect of temperature on development in wolffish: comparison with salmon

Peculiarities of reproduction, early ontogeny, methods of egg incubation, and influence of temperature on development are compared in wolffish (Anarhichas lupus L.) and in salmonids, mainly Atlantic salmon (Salmo salar L.). Reproductive indices of females are similar in both species, but differ in males due to the small production of sperm in wolffish, a difference explained by internal fertilization in the latter species. The rate of embryonal development until the beginning of differentiation of fin fold at the same temperature is similar in both species, but the period from activation to hatching is more than twice as long in wolffish as in Atlantic salmon. Wolffish hatch at a more advanced stage with a small remnant of yolk sac and begin to feed shortly thereafter. As in Atlantic salmon, eggs and larvae of wolffish have a wide range of temperature resistance except before and after hatching. Based on the difference of life cycles in wolffish and salmon, technologies for their artificial breeding are compared.     

Cryopreservation of carp sperm

About 70% of eggs of Cyprinus carpio reached the eyed stage in fertility tests with 1 ml of cryopreserved semen added to 1 g of eggs. When 1 ml of semen was added to 100 g of eggs, few eggs reached the eyed stage. In two fertility tests with sperm preserved for 342 days, using 5 g of eggs inseminated with 1 ml of semen, percentages of eyed eggs were 31.5 and 25.5, respectively. In a series of fertility tests, the chemical constituency of the medium in which fertilization was attempted influenced the fertility of cryopreserved sperm.     

Growth and digestibility effects of soya lecithin and choline chloride on juvenile Atlantic salmon

Atlantic salmon, Salmo salar L., fingerlings with a mean weight of 1.5 g were fed one of four casein-gelatine-based purified diets supplemented with soya lecithin (LC) and choline chloride (CH) for 84 days. The diets were supplemented with either: 0 g kg⁻¹ CH and 0 g kg⁻¹ LC, 0 g kg⁻¹ CH and 30 g kg⁻¹ LC, or 5 g kg⁻¹ CH and 0 g kg⁻¹ LC, or 5 g kg⁻¹ CH and 30 g kg⁻¹ LC. The same diets were also fed to 100-g salmon to assess the effects of LC and CH supplementation on digestibility. Fingerlings fed the diet with neither LC nor CH (0 g kg⁻¹ LC and 0 g kg⁻¹ CH) grew at a significantly slower rate than fish fed the supplemented diets. There were significant effects on growth of supplementation of both LC and CH. The results indicate that the choline requirement of Atlantic salmon fingerlings is satisfied by 4 g kg⁻¹ inclusion in a gelatine-casein-based diet, and that dietary soya lecithin can fully replace choline chloride. The digestibility study with the larger fish indicated a beneficial effect of lecithin on the digestibility of both protein and energy.     

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.     

Creation of artificial upwelling areas for brown trout, Salmo trutta, spawning in still water bodies

Abstract  Brown trout, Salmo trutta L., spawning sites were constructed by creating areas of artificial upwelling water, 252 ± 37 mL m⁻² min⁻¹ (95% CL), through appropriately sized spawning gravel substrate in 3 m² vessels buried in the bottom of a 150-m² pond. Natural spawning occurred in the vessels during autumn 2001–2004, with hatching and alevin swim up the following spring. In areas of upwelling, egg survival was 85–95%, while no live eggs were observed in areas without upwelling. In areas with upwelling, the maximum density of live eggs at the eyed stage was 570–1510 eggs m⁻². In spring 2004 and 2005, the density of alevins was estimated at 322 (±187) m⁻² and 567 (±217) m⁻², respectively, in areas with upwelling water, compared with 35.2 ± 25.4 m⁻² in areas without upwelling water in 2004.     

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.     

Inter-river, -annual and -seasonal variability in fecundity of Atlantic salmon, Salmo salar L., in rivers in Newfoundland and Labrador, Canada

Abstract  Fecundity is an integral component of the calculation of Atlantic salmon, Salmo salar L., egg depositions in rivers. Fecundity determinations can be time consuming and prohibitively expensive in terms of application on a broad scale. Consequently, where river specific and annual data are not available, default means are used in calculations in Newfoundland and Labrador. It is important therefore to know the extent of variability among rivers, years and seasons and the potential error involved in using default values. Annual fecundity data were available for one river in Labrador and nine rivers in Newfoundland. Fecundity was determined from ovaries collected in the recreational fishery in the summer for all 10 rivers. For three of these rivers, fecundity determined from summer sampling was compared with that obtained from sampling at time of spawning in autumn. There was significant variability in fecundity with length as a covariate among rivers, years and seasons. Mean number of eggs per female decreased between 8.3% and 29.0% from summer to autumn while mean number of eggs per cm decreased from 5.0% to 28.5%. Depending on the measure of relative fecundity used (no. of eggs kg⁻¹ or no. of eggs cm⁻¹), results of simulations showed that estimates of egg deposition incorporating defaults can deviate from those obtained by applying year-specific and river-specific values by 50–75%, without adjusting for the seasonal reduction in fecundity, and by 30–50% with an adjustment. A sensitivity analysis revealed that of three parameters used in the calculation of egg deposition (size, percent female and fecundity), fecundity was the most influential.     

Artificial reconditioning, spawning and survival of Atlantic salmon, Salmo salar L., kelts in salt water and survival of their Fl progeny

Abstract. Atlantic salmon kelts were successfully reconditioned in salinities of 16% and 28%o using a moist pellet diet. Two groups of reconditioned kelts were re-acclimated to fresh water in the autumn tu prevent super chitling in salt water during the winter. The feeding response and growth of salmon kelis. both years, was highest when salinities were maintained at 28% during the winter and 16%during the summer. However, other salinities also provided acceptable growth rates. Two-sea-winter salmon kelts reconditioned at a slower rate in all groups than 1-sea-winter salmon kelts. Kelt survival was 93.1% the first year and 82.7% the second year. Gonnd maturation occurred in 17.9% of the female 1-sea-winter kelts and 40.6% of the male 1-sea-winter kelts after the first year. At the end of the second year. 83.3% of the 1-sea-winter female kelts and 96.3% of the l-sea-winter male kelts matured and spawned. Reconditioned salmon kelts with a mean fork length of 68.4 cm produced 7261 eggs per fish with a mean egg diameter of 5.70 mm and a fecundity of 2262 eggs.kg⁻¹, Survival (Fl) from the green egg stage to the alevin and smolt stages was 73.5% and 38.8% respectively. The smolt to adult return rule based on unadjusted tug recaptures was 2.20%.     

The Induction of Tetraploidy in Salmonids

The induction of tetraploidy was attempted in three intraspecies crosses (autotetraploid) of sal-monid: chinook salmon ( Oncorhynchus tshawytscha ), coho salmon ( O. kisutch ) and rainbow trout ( Salmo gairdneri ), and in the interspecies hybrid (allotetraploid) coho × Atlantic salmon ( Salmo salar ).
Pressure treatments of 6.2 × 10⁴ kPa (9,000 psi) were applied for six minutes at intervals from 50% of the time to cleavage to beyond first cleavage. Analysis of eyed stage embryos via flow cytometry indicated two periods of tetraploid induction existed corresponding to 55–75% and 100–110% of the first cleavage interval (FCI).
The effects of different pressures and the times of application were analyzed in a study with rainbow trout. Treatments of 4.8 × 10⁴ kPa (7,000 psi) for eight minutes, and 5.5 and 6.2 × 10⁴ kPa (8,000 and 9,000 psi) for four minutes, were applied at 65, 78, and 100% FCI. Tetraploids were found in the 5.5 and 6.2 × 10⁴ kPa group treatments only when applied at 65% FCI.
The results indicate that, while tetraploidy is inducible in salmonids, further experimentation is necessary to discern the appropriate time and treatment for optimal tetraploid induction in each species.     

Artificial fertilization in turbot, Scophthalmus maximus (L.): different methods and determination of the optimal sperm–egg ratio

With the aim of improving artificial fertilization (AF) in turbot, Scophthalmus maximus (L.), a series of fertilization experiments was carried out under dry conditions and different wet conditions (eggs/sea water: 2V/V and V/V). Another series of fertilization experiments was carried out with different quantities of sperm pool to determine the optimal ratio of spermatozoa to eggs for each AF method. Sperm pool from two males and eggs from spawns with a viability rate of > 70% were used. The sperm pool’s density (0.4–5.18 × 10⁹ sperm mL^–1) and motility (1–5) had been assessed previously. Significantly different fertilization rates were found when comparing 2V/V and V/V wet conditions. Significantly higher fertilization rates were found in dry fertilization when the sperm–egg ratio was > 9000 spermatozoa per egg and, under wet condition V/V, at 3000–4000 spermatozoa per egg.     

Effects of Environmental Sodium Chloride on Percent Hatch, Yolk Utilization, and Survival of Channel Catfish Fry

Abstract.— Only limited research has addressed the effect of salinity on hatching of channel catfish Ictalurus 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 1 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 NaSO₃ significantly reduced percent hatch at all NaCl levels. Although our results indicate that addition of NaCI 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.     

Production of market-size European strain Atlantic salmon (Salmo salar) in land-based freshwater closed containment aquaculture systems

Interest in land-based farms using recirculating aquaculture systems (RAS) for market-size Atlantic salmon (Salmo salar) continues to grow, and several commercial facilities are already rearing fish. Performance data for commercially available mixed-sex, all-female, and triploid all-female Atlantic salmon reared to market-size in freshwater land-based facilities, however, are limited, particularly for European strain fish. Accordingly, eight groups of European-sourced Atlantic salmon (five groups of diploid mixed-sex, two groups of diploid all-female, and one group of triploid all-female fish) were reared from eyed egg to market-size in a semi-commercial scale land-based aquaculture systems over five separate production cycles to quantify performance metrics. Fish reached market-size (4−5 kg) in 24.7–26.3 months post-hatch. Fish were reared at a mean water temperature of 12.3–13.7 °C from first feeding to a mean size of 466–1265 g, then 13.3–15.1 °C during growout. On average, all-female groups grew faster than mixed-sex groups; however, environmental conditions and performance of individual cohorts varied. In a comingled production cycle, diploid all-female salmon grew faster than triploid counterparts. Early maturation rates ranged from 0 % to 67 %, with a mean maturation rate of 34 % for diploid mixed-sex fish and 67 % and 13 % for two diploid all-female groups, respectively. Triploid all-female Atlantic salmon did not mature. This research confirms biological and technological feasibility of growing Atlantic salmon to market-size in land-based systems but controlling early maturation of diploid salmon remained a challenge under the conditions utilized in these trials. This research provides important data inputs to optimize operational and financial projections for existing and potential land-based Atlantic salmon farms.