Effects of light spectrum on growth and physiological status of gilthead seabream Sparus aurata and rainbow trout Oncorhynchus mykiss reared under recirculating system conditions

Previously reported data clearly indicate that depending on species, ambient light spectrum can affect fish growth, physiology, behaviour, reproduction, etc. Since light spectrum can be easily controlled in intensive indoor fish farming facilities, the present study aimed to investigate the effects of coloured light on growth performance (body weight, growth rate, food utilization, carcass composition, etc.) and physiological status (blood and plasma parameters, brain neurotransmitters, tissue fatty acid composition, etc.) of two widely reared fish species, gilthead seabream Sparus aurata and rainbow trout Oncorhynchus mykiss. For this purpose, 240 specimens of S. aurata (29.8 ± 0.13 g) and 60 specimens of O. mykiss (71.4 ± 0.30 g) were exposed to white (full-spectrum, fluorescent lamps), red (605 nm) and blue (480 nm) light (lamps covered with appropriate filters) for 11 weeks under recirculating water systems. Blue light had a significantly negative impact on O. mykiss growth performance accompanied with reduced liver total lipids and plasma glucose and increased brain serotonergic and dopaminergic activity. In the case of S. aurata, red light significantly increased brain dopaminergic activity, while a tendency towards reduced growth was also observed. Since these results indicated the establishment of stressful conditions, O. mykiss and S. aurata should not be reared under blue and red light, respectively. For each species, the effect of the remaining light colours tested, was not clearly differentiated so that an appropriate light spectrum for the most efficient farming of O. mykiss and S. aurata could not be suggested, at least for the time period examined. Nevertheless, present results suggest that light colour should be regarded as a rearing factor worth to be further investigated, especially when recirculating systems are concerned.     

The development of a sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum): evaluation of cryoprotectants and diluents

Three cryoprotectants (dimethyl sulphoxide, propylene glycol and glycerol) and two diluents (sucrose based and saline based) were mixed (9 parts diluent–1 part cryoprotectant) factorially to produce six extenders that were tested to develop an effective sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum). Sperm were diluted 1:3 with each extender and frozen by flotation on liquid nitrogen before being submerged and stored for 30 days. Sperm left unfrozen in each extender for 20 min showed no toxic effects on motility. Extenders containing propylene glycol (PG) as cryoprotectant yielded higher post‐thaw sperm motilities than those containing dimethyl sulphoxide (DMSO) or glycerol. The sucrose‐based diluent performed better than the saline‐based diluent when DMSO was used as cryoprotectant, but there were no differences in post‐thaw motility between diluents for the other cryoprotectants. Activating sperm with ovarian fluid and sea water instead of sea water alone had no effect on post‐thaw motility. In fertilization trials, no differences were observed between any of the extenders and fresh milt when milt, eggs and sea water were left in contact for 1 h. When sperm were forced to compete for eggs by reducing contact time to 20 s, fertilization results followed those of sperm motility rates. Percentage hatch and morphology of larvae at hatching did not differ for eggs fertilized by cryopreserved and fresh sperm. This study represents the first reported successful attempt at cryopreserving winter flounder sperm and should improve gamete and broodstock management protocols for this species.     

Two microalgae Crypthecodinium cohnii and Phaeodactylum tricornutum as alternative source of essential fatty acids in starter feeds for seabream (Sparus aurata)

Despite oils extracted from algae and other microorganisms that may constitute excellent sources of HUFAs, few studies have determined the nutritional value of different microalgal species for young marine fish. Six thousand gilthead seabream (Sparus aurata) postlarvae (73 mg body weight) were fed for 57 days diets containing either fish oil as a single lipid source or 2 and 4% of Cryptecodinum cohnii or 5% Phaeodactylum tricornutum. Fish oil substitution by C. cohnii resulted in improved fish survival and a very good growth performance, in agreement with a higher proportion of DHA in diets and in total lipids of fish. Incorporation of DHA and other fatty acids was proportional to their contents in diet suggesting the good nutritional utilization of homogenized C. cohnii. Lower survival rates were found in fish fed P. tricornutum and could be related to an epithelial degeneration observed in the anterior intestine. This degeneration could be related to a higher lipid content in these fish or to the strong hornlike cornutate processes found in the valves of the diatom P. tricornutum.     

The effects of osmolality on sperm quality in <Emphasis Type="Italic">Jenynsia multidentata</Emphasis> (Cyprinodontiformes: Anablepidae)

Sperm quality tests on fish are classically used for evaluating cryopreservation procedures, and they are also promising to assess aquatic toxicity and biomarkers of xenobiotic effects on reproduction. Osmotic shock from the storage medium is one of the main factors affecting sperm quality during evaluation. Thus, the objective of this study was to evaluate the effects of different osmolalities (240–460 mOsm/kg) for at least 4 days on the sperm quality parameters of the viviparous fish Jenynsia multidentata. The level of significance was (P < 0.05). The plasma osmolality of J. multidentata is 326 ± 3.9 mOsm/kg. The motility of fresh semen was higher in osmolalities of 280 and 300 mOsm/kg but did not differ between osmolalities from 240 to 320 mOsm/kg. Above 380 mOsm/kg, the motility observed was 0 %. Over the time period studied motility increased with increasing osmolality, and the most constant and long-lasting rates were between 300 and 320 mOsm/kg. On the 4th day of evaluation, higher membrane integrity rates were observed between 280 and 360 mOsm/kg, higher mitochondrial membrane potential was observed between 300 and 460 mOsm/kg, and higher DNA integrity rates were observed between 260 and 380 mOsm/kg. Moreover, osmolalities ≥460 and ≤240 resulted in the lowest motility and DNA integrity levels. Over 4 days, the plasma membrane integrity was significantly lower at ≤260 and ≥400 mOsm/kg, and the mitochondrial membrane potential was significantly lower only in osmolalities ≤240 mOsm/kg. Therefore, we conclude that for sperm quality preservation in J. multidentata, an osmolality of 300–320 mOsm/kg of the most suitable diluent is necessary. Furthermore, we conclude that the storage of sperm in a hyposmotic (<260 mOsm/kg) or hyperosmotic (>400 mOsm/kg) solution affects not only motility but also other sperm quality parameters.     

Ovarian fluid pH enhances motility parameters of rainbow trout (Oncorhynchus mykiss) spermatozoa

All evidence to date suggest that sperm motility is the primary determinant of fertilization success in externally fertilizing fish species. Ovarian fluid, which comprises 10–30% of the total egg volume in salmonids, enhances sperm motility with respect to swimming speed, trajectory and the duration of movement. It was recently demonstrated that there is individual variability in sperm motility enhancing potential of ovarian fluid of particular females. In the present study we examined the effect of particular ovarian fluids collected from 31 females on the sperm motility parameters of one male of rainbow trout (Oncorhynchus mykiss) using computer-assisted sperm analysis (CASA). During our experiment we also monitored the pH of ovarian fluid. We found that particular fluids differed in the ability to activate spermatozoa; sperm remained immotile in four fluids and exhibited 50–100% motility in 27 samples. The percentage of motile sperm, velocity and duration of movement positively correlated with ovarian fluid pH (r² = 0.34–0.62). These data strongly suggest that the pH of the ovarian fluid is the primary determinant of sperm motility in rainbow trout under natural conditions of fertilization.     

Comparative study of digestive enzymes in fish with different nutritional habits. Proteolytic and amylase activities

This work provides a comparative study of the proteolytic and amylase activities in six species of fish with different nutritional habits: rainbow trout (Oncorhynchus mykiss), gilthead seabream (Sparus aurata), European eel (Anguilla anguilla), common carp (Cyprinus carpio), goldfish (Carassius auratus), and tench (Tinca tinca). Trout and carp showed the highest digestive proteolytic activity. When proteolytic activity was determined in a wide range of pHs, the highest values in the digestive tract of all species were found at alkaline pHs, except in eel where activity could be detected only at acid pHs. Eel showed the lowest digestive proteolytic potential among all the species studied. With respect to amylase activity, the omnivorous species presented higher activity than did the carnivores. Among the carnivorous species, the lowest activity was found in trout. The ratio of total amylase:total proteolytic activity was higher in omnivorous fish species, the carp having the greatest value, whereas in trout this ratio was lower than one. Digestive enzyme activity declined as the incubation temperature decreased, but this trend varied depending on the fish species and the tissue analyzed.     

Extenders and cryoprotectants for cooling and freezing of piracanjuba (Brycon orbignyanus) semen, an endangered Brazilian teleost fish

The aim of this study was to develop a protocol for semen storage of piracanjuba (Brycon orbignyanus) by both cool storage at 4 °C and cryopreservation at − 196 °C. Semen was diluted in some fish semen extenders (Exp. 1) or in extenders combined with the antibiotic gentamycin sulfate (Exp. 2) and stored at 4 °C. Sperm motility was estimated every 24 h. Then, the effects of egg yolk (0 and 5%), cryoprotectants (dimethyl sulphoxide — DMSO, methanol, and methylglycol) and extenders (NaCl 154 mM, BTS™ Minitub and M III™ Minitub) on semen cryopreservation were evaluated (Exp. 3). Semen was added to each of eighteen cryosolutions (2 yolk concentrations × 3 cryoprotectants × 3 extenders), aspirated into 0.5-mL straws, frozen in nitrogen vapor (Taylor-Wharton, CP 300, “dry shipper”) and stored at − 196 °C. Sperm motility was evaluated after thawing at 60 °C-water bath for 8 s. The three cryosolutions that produced the highest post-thaw sperm motility were used again to freeze semen. Post-thaw semen quality was then evaluated under three tests: sperm motility, the percentage of live spermatozoa and hatching rate (Exp. 4). Piracanjuba semen diluted (1:10 total volume) in NaCl 200 mM or in Saad solution (NaCl 200 mM, Tris 30 mM) maintained motility above 35% for as long as 7 days, at 4 °C. Motility of only 7% was observed on undiluted semen after 3 days at 4 °C. There was neither beneficial nor detrimental effect of gentamycin on sperm motility at 250 μg/mL. Egg yolk addition to the cryosolution was beneficial in samples cryopreserved in NaCl 154 mM and in M III™, but detrimental for samples cryopreserved in BTS™. Methylglycol was the most effective cryoprotector compared to DMSO and methanol. Motility and percentage of live spermatozoa were similar among semen cryopreserved in NaCl–yolk, M III™–yolk and BTS™, all containing 10% methylglycol, but lower than fresh control. Hatching rates of eggs fertilized with sperm cryopreserved in NaCl–yolk or BTS™ were higher than for eggs fertilized with sperm cryopreserved in M III™–yolk, but lower than control fertilizations. The semen cryopreservation protocols developed here will be used to set up a gene bank for endangered piracanjuba populations.     

Flumequine depletion from muscle plus skin tissue of gilthead seabream (Sparus aurata L.) fed flumequine medicated feed in seawater at 18 and 24 °C

We examined flumequine depletion from muscle plus skin of gilthead seabream held in seawater at 18 and 24 °C. Seven groups of 10 fish each were sampled at intervals ranging from 24 to 168 h after in-feed administration of flumequine at 35 mg/kg/day for 5 days. Muscle plus skin tissue samples were analyzed for flumequine by high-performance liquid chromatography and fluorescence detection (HPLC-SFD). Parent flumequine concentrations declined rapidly from muscle plus skin after dosing with elimination half-lives of t_1/2=22.14 and 21.43 h at 18 and 24 °C, respectively. Withdrawal periods for the maximum residue limit (MRL) of 600 μg/kg flumequine in muscle plus skin at 95% tolerance limit were 106.08 and 75.84 h at 18 and 24 °C, respectively, after treatment.     

Refrigerated storage and cryopreservation of sperm of red drum, Sciaenops ocellatus L.

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 539 ± 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 post-thaw 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.     

Changes in Atlantic cod (Gadus morhua L.) sperm quality during the spawning season

The biology of cod reproduction is well described in the scientific literature. However, sperm biology and spermatozoa management are poorly studied in this species. Because of its recent farming expansion, a better knowledge of cod gametes is becoming especially useful. This work aimed at establishing tools to study sperm biology in cod, and also investigated the existence of changes in cod sperm quality during the spawning period. We showed that sperm concentration could be assessed using spectrophotometry at 260 nm. Sperm motility significantly decreased after a 168‐h storage at 4 °C. A 1:9 dilution of sperm in a non‐activating medium (1/3 seawater and 2/3 freshwater, osmotic pressure: 360 mOsm kg^?1) improved sperm storage. Sperm concentration, sperm velocity and storage capacity at 4 °C peaked during the medium period of the spawning season and then decreased to values close to those observed at the beginning of the reproductive period. The measured values of osmotic pressure, pH, protein, Na⁺, Cl^? and Ca²⁺ concentrations of the seminal fluid were modified along the spawning period. Cell damage was noted at the end of the spawning period: local blebs were observed on the flagellum but also loops at its distal part. On the other hand, spermatocrit did not vary with the sampling date. In conclusion, cod sperm quality is modified during the spawning period, the highest‐quality samples being collected during the medium part of this season.     

Spawning of ocean pout (Macrozoarces americanus L.): evidence in favour of internal fertilization of eggs

Sperm physiology, in vivo artificial insemination and spawning of the ocean pout (Macrozoarces americanus L.), a marine bottom fish, were studied. Milt was collected from the reproductive tract of mature males by suction using a catheter. The uncontaminated milt, having a very low sperm concentration, contains highly motile spermatozoa and sperm motility was retained in vitro at 4 °C for at least 24 h in both seminal plasma and ovarian slime collected from the oviduct of pre-spawning females. Instead of activating sperm, dilution in sea water instantly immobilized the spermatozoa of ocean pout. Osmolarity and pH of ocean pout seminal plasma were in the ranges 365–406 mOsM and 7.2–7.5, respectively. A study of the ionic composition of ocean pout seminal plasma demonstrated the presence of various ions including Na⁺, K⁺, Ca²⁺, Mg²⁺, and Cl⁻, with a remarkably lower K⁺ concentration compared to that from other fish species. Since injections of milt containing motile sperm into the ovaries of pre-spawning females, which spawned in the absence of males, yielded fertilized ocean pout eggs, it is concluded that the ocean pout exhibits internal fertilization. The larvae hatched after 3 months of egg incubation in ambient sea water (9–10 °C). With proper timing of in vivo artificial insemination of mature females, fertilized ocean pout eggs can be obtained from fish reared in captivity.     

Cryopreservation diluents for spermatozoa of Sakhalin taimen <Emphasis Type="Italic">Hucho perryi</Emphasis>

To develop a suitable cryopreservation diluent for spermatozoa of the endangered Sakhalin taimen Hucho perryi, all possible combinations of cryoprotectants (glycerol, dimethyl sulfoxide [DMSO], methanol) and extenders (fetal bovine serum [FBS], 300 mM glucose solution [GS], artificial seminal plasma for masu salmon) were examined by observing sperm motility 10 s after thawing. Spermatozoa cryopreserved with diluents such as mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS showed the highest motility. The maximal post-thaw motility was observed at 10% among all concentrations (0, 5, 10, 15 and 20%) of these three cryoprotectants. No significant difference among three diluents was observed in motility at 10 s. Mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS are suitable cryopreservation diluents for Sakhalin taimen spermatozoa.     

Abnormalities of the operculum in gilthead sea bream (Sparus aurata): morphological description

The authors studied a population of 582 gilthead sea breams (Sparus aurata), aged 25–600 days, in order to describe the abnormalities of the opercular complex.

Particular attention was paid to: (1) the different degrees of abnormality of the opercular complex as a whole and of the individual dermal bones (preopercle, interopercle, subopercle and opercle) that compose it; (2) the ways in which the operculum folds into the gill cavity and the relevant histological characteristics. This study discusses the origin and nature of the abnormalities.

Though these abnormalities have a major impact on market value and product image, and are often linked to other disorders in fish, they are still difficult to eliminate. Gaining a basic understanding of these abnormalities will be a first step towards reducing their occurrence.     

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 .     

Reproductive performance of gilthead seabream (Sparus aurata L., 1758) fed two combined levels of carotenoids from paprika oleoresin and essential fatty acids

Despite carotenoids and essential fatty acids seem to play important roles in fish reproduction, no studies have yet been conducted to determine the effect of dietary carotenoids on gilthead seabream broodstock performance and their relation as antioxidants with dietary n‐3 highly unsaturated fatty acid (HUFA) levels. In addition, the high cost of synthetic sources of carotenoids is leading to the search for new natural carotenoid sources such as paprika oleoresin. Four experimental diets containing two combined levels of carotenoids from paprika oleoresin (40 and 60 mg kg^?1) and n‐3 HUFA (25 and 40 g kg^?1) were respectively fed to triplicate groups of gilthead seabream (Sparus aurata) broodfish. Elevation of n‐3 HUFA dietary levels from 25 to 40 g kg^?1 significantly improved gilthead seabream broodstock performance in terms of egg viability, hatching rates and fecundity. Besides, it markedly increased egg contents in HUFAs which play important energetic and structural roles and improve embryo development. Both arachidonic acid and eicosapentaenoic acid (20:5n‐3) egg contents were more readily affected by dietary n‐3 HUFA than docosahexaenoic acid. HUFA levels did not caused any negative effect suggesting an optimized content of antioxidants in broodstock diets. Increase in dietary carotenoids from 40 to 60 mg kg^?1 increased carotenoid contents in eggs and significantly improved egg fertilization rates suggesting an important sperm cell’s protective role by reducing the risk of lipid peroxidation which is detrimental for sperm motility. The increased inclusion of dietary paprika oleoresin enhanced egg carotenoid deposition and improved fish reproductive performance, denoting the high nutritional value of this product as a source of carotenoids for broodstock of this species.     

An Efficient Methodology for Cryopreservation of Spermatozoa of Red Seabream, Pagrus major, with 2-mL Cryovials

The aim of this study was to optimize the cryopreservation protocols for the sperm of red seabream, Pagrus major. The 2‐mL cryovials and programmable freezer were employed for cryopreservation. Six extenders, six cryoprotectants in various concentrations ranging from 6 to 20% (v/v), four cooling rates, and three thawing temperatures were evaluated by postthaw sperm motility and fertility. The ratio of sperm to egg for postthaw sperm fertilization trials was experimentally standardized and was optimal at 500:1. The best motility of postthaw sperm (79.4 ± 4.7% to 88.6 ± 8.0%), fertilization rates (89.6 ± 2.9 to 95.6 ± 1.9%), and hatching rates (85.3 ± 5.1% to 91.4 ± 4.3%) were achieved when Cortland extender, dimethyl sulfoxide (15, 18, and 20%) or ethylene glycol (9, 12%) as cryoprotectants, 20 C/min as the cooling rate, and 40 C as the thawing temperature were employed. Moreover, the results on embryonic development were not significantly different between cryopreserved sperm and fresh sperm during incubation process. In conclusion, these methods of cryopreservation of red seabream sperm are suitable for routine aquaculture application and preservation of genetic resources.     

Filtration of king scallops (Pecten maximus)

Filtration rates of hatchery-reared king scallop (Pecten maximus L.) juveniles, fed a single species alga diet (Pavlova lutheri (Droop) Green), were measured at a range of temperatures (6–21 °C). Weight specific filtration rate (ml min⁻¹ g⁻¹ (live weight)) of juveniles of a selected size range of 17–19 mm shell height (0.26–0.36 g live weight) increased with temperature above 16 °C and decreased below 11 °C, but was not significantly different between these two temperatures. Measurements at 16 °C using juveniles with a wider size range of 10–25 mm shell height (0.05–0.8 g live weight) gave the allometric equation: filtration rate (ml min⁻¹)=12.19×weight (g)^0.887. Filtration rate decreased significantly when the cell concentration was greater than 200 cells μl⁻¹ (4.25 mg (organic weight) l⁻¹). With six other algae food species, filtration rates similar to those with P. lutheri were only achieved with Chaetoceros calcitrans (Paulsen) Takano. All other algae species tested were cleared from suspension at significantly lower rates. Experiments with diet mixtures of P. lutheri and these other algae suggested that this was usually a reflection of lowered filtration activity, rather than pre-ingestive rejection of cells. In experimental outdoor nursery rearing systems, the filtration rate was inversely proportional to the concentration of cells in the inflow, in the range 5–210 cells μl⁻¹. It was not affected by flow rate (2–130 l h⁻¹, equivalent to 0.12–28.38 l h⁻¹ g⁻¹ (live weight)) with scallop juveniles stocked from 2 to 62 g l⁻¹. The results are discussed in relation to on-growing scallops at field sites.     

Sublethal effects and safe levels of carbon dioxide in seawater for Atlantic salmon postsmolts (Salmo salar L.): ion regulation and growth

Atlantic salmon (Salmo salar L.) postsmolts (0.17–0.26 kg) were exposed to four different levels of carbon dioxide partial pressure for 43 days in an open flow system: 0.6 mm Hg (control), 4.9 mm Hg (low), 12 mm Hg (medium), and 20 mm Hg (high). The water temperature was 15–16°C and the salinity 34‰. In the low carbon dioxide group (P_CO2=4.9 mm Hg; 10.6 mg/l), no significant differences were found in blood parameters (haematocrit, plasma chloride and plasma sodium) or in growth parameters (weight, length and condition factor) when compared to the control group. After 43 days, the mean plasma chloride concentration for the medium group (P_CO2=12 mm Hg; 26 mg/l) was significantly reduced, while weight and condition factor were slightly, although not significantly, lowered. For the high carbon dioxide group (P_CO2=20 mm Hg; 44 mg/l) plasma sodium and plasma pH were significantly increased and plasma chloride, oxygen consumption, weight, length and condition factor were significantly reduced at the end of exposure. There was no mortality in the control group or in the low carbon dioxide group. The mortalities in the medium and high carbon dioxide groups were 1.1 and 4.3%, respectively. Nephrocalsinosis was not observed in any of the groups. The results of the present investigation indicate that the CO₂ concentration of the low group may represent a safe level for Atlantic salmon postsmolts when the temperature is 15–16°C and the oxygen level is 6–7 mg/l. Further studies are required.     

Effect of extender composition and freezing rate on post-thaw motility and fertility of Arctic char, Salvelinus alpinus (L.), spermatozoa

The effects of extender composition and freezing rate on motility and fertility of frozen‐thawed Arctic char, Salvelinus alpinus, spermatozoa were investigated. Three freezing rates, two semen diluents and three cryoprotectants were tested. Semen frozen in 0.3 mol L^?1 glucose diluent with 10% methanol as a cryoprotectant or in a diluent described by Lahnsteiner with 10%N,N‐dimethylacetamide (DMA) resulted in the highest sperm motility. Fertility was the highest for semen frozen in a glucose–methanol extender but was not significantly different than that for semen frozen in Lahnsteiner's diluent with 10% DMA. Dimethyl sulphoxide (DMSO) at 10% was a relatively ineffective cryoprotectant with either semen diluent. Semen frozen at 6 cm above the surface of liquid nitrogen resulted in a higher post‐thaw sperm motility and fertility than semen frozen at 5 cm. The addition of 7% fresh egg yolk to glucose diluent containing methanol or DMSO did not improve the fertility of frozen‐thawed spermatozoa. However, the addition of 7% fresh egg yolk to glucose–DMA extender significantly improved the fertilization percentages of frozen‐thawed spermatozoa. In conclusion, dilution of semen 1:3 in 0.3 mol L^?1 glucose with 10% methanol and freezing 6 cm above the surface of liquid nitrogen (freezing rate of 40±8°C min^?1, mean±SD from ?5 to ?55°C) is a promising protocol for cryopreservation of Arctic char semen.     

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.