Effect of temperature on short-term storage of eggs and sperm of chum salmon (Oncorhynchus keta)

Chum salmon (Oncorhynchus keta) gametes were stored separately for intervals up to 460 h at temperatures ranging from 3 to 15°C. Estimates of time to 90 and 50% fertilization success (FS) were obtained for either stored eggs, stored milt, or stored eggs and milt. When both gametes were stored, the combined loss of gamete viability resulted in shorter times to 90 and 50% FS than when either eggs or milt were stored alone. Gamete storage at colder temperatures significantly prolonged viability; for example, when both gametes were stored, the time to 90% FS ranged from 19.6 h (15°C) to 123.9 h (3°C). Loss of viability of stored gametes in these trials was similar to that found in other studies with Oncorhynchus species at low storage temperatures (3–6°C), but gametes showed significantly greater viability when stored at higher temperatures (9–15°C). This increased viability in the current study appears to have resulted from the provision of (1) higher air-to-gamete ratios, and (2) greater interface area between air and gametes in the storage containers, two factors that would enhance gamete respiration during storage.     

Short-Term Storage of Semen of Rainbow Trout: Interactions of Time,Antibiotic, and Activator

This study was designed to examine the main effects and interactions of time, presence of antibiotics, and type of sperm activators on the fertilization capacity (eyeing rate) of refrigerated semen of rainbow trout, Oncorhynchus mykiss. The semen samples were stored in the presence or absence of 250 IU ml^?1 penicillin and 250 μg ml^?1 streptomycin sulfate. Freshwater and a saline solution were used as sperm activators. The semen samples were stored at 2–3°C and fertilized after 0, 6, 8, 12, 19, and 25 days of storage. Fertilizing capacities of semen samples stored in the presence of antibiotics (63.8 ± 5.6%) were significantly (p < 0.05) higher than those stored in the absence of antibiotics (46.2 ± 6.7%). Also, the fertilizing capacities of stored semen samples activated using saline solution (70.7 ± 5.7%) had significantly (p < 0.05) higher values than those activated using freshwater (39.3 ± 5.9%). Semen samples stored in the absence of antibiotics completely lost fertilizing capacity within 19 days of storage. After 25 days of storage in the presence of antibiotics, induction of fertilization using freshwater and saline solution resulted in 0% and 79.8 ± 1.7% fertility, respectively.     

Computer-controlled freezing of rainbow trout Oncorhynchus mykiss (Walbaum) spermatozoa for routine programmes

The effects of extender composition and freezing rate on cryopreservation efficiency of refrigerated spermatozoa of rainbow trout Oncorhynchus mykiss (Walbaum) were evaluated in order to test the suitability of a computer-controlled ultrafreezer to cryopreserve milt samples obtained in field conditions and stored for several hours. A very highly significant first-order interaction between freezing rate and the type of extender was found. Six of the eight experimental variants did not differ significantly, resulting, after fertilization of eggs with cryopreserved sperm, in a range of 62.3–74.8% of eyed embryos. This procedure was effective for samples stored at 1 °C for 2 days.     

The chemical and sensorial changes in rainbow trout caviar salted in different ratios during storage

In this study, the intention was to investigate the chemical and sensorial changes occurring during cold storage (4 ± 1°C) of caviar obtained from rainbow trout Oncorhynchus mykiss W. 1792 that was dry salted in different ratios. Caviar has a higher fat and protein content than other seafood products, and in processing it with dry salting technology, it was determined that the shelf life could be up to 28 days for the experimental group in which salt was applied at a rate of 4% and up to 35 days for the experimental group in which salt was applied at a rate of 8%. Thus the storage period of the samples is longer for the products to which a high (8%) amount of salt was applied than the products to which a low (4%) amount of salt was applied in the salting process.     

Improving cold storage of subitaneous eggs of the copepod Acartia tonsa Dana from the Gulf of Mexico (Florida – USA)

Developing methods to store copepod eggs is necessary to increase the availability of copepods as a live food for the aquaculture industry and aquarium trade, and also to allow the exchange of copepods between researchers. The present study, evaluated the effect of glucose and two antibiotics (kanamycin sulphate and oxytetracycline HCl) on extending the shelf life of cold-stored subitaneous Acartia tonsa eggs. Also, egg development effects on the survival of the eggs were tested. Glucose did not have any significant effects on the survival of the eggs. However, the addition of antibiotics to the storage vials resulted in an increase of the survival of the eggs. In the best case, the shelf life of the eggs was almost doubled. After 7 days, the kanamycin+glucose treatment led to a hatching success of 86±1% of the hatchable eggs, while the untreated eggs presented a hatching success of 47±6%. However, long exposure to high concentrations of antibiotics was lethal to the copepod eggs. After more than 30 days of exposure to 100 mg L⁻¹ of oxytetracycline, the survival of the eggs was lower than in the untreated samples. After 45 days, oxytetracycline-treated eggs (100 mg L⁻¹) presented a hatching success of 4–5% while the non-stored eggs still had a hatching success of 9%, and the eggs treated with a lower concentration of antibiotics (10 mg L⁻¹) showed a hatching success up to 21–23%. The size of the nauplii in all trials tended to decrease as the period of cold storage at 1°C increased. We consider that the use of antibiotics at the right dosage to be a means to increase the storage capacity of the Gulf of Mexico strain of A. tonsa eggs, which do not show any capacity to be stored for long periods of time, compared with some other strains. In addition eggs that were between 5 and 7 h old survived longer when stored in the cold than eggs, which were freshly spawned or closer to hatching.     

Effects of storage and incubation temperature on the viability of eggs, embryos and larvae in two strains of an African catfish, Heterobranchus longifilis (Siluriformes, Clariidae)

Two experiments, dealing with short‐term storage of ova and thermal conditions to optimize gamete and eggs management in hatcheries of the African catfish, Heterobranchus longifilis, were carried out. In the first experiment, ova collected by stripping from two strains of H. longifilis were stored for intervals up to 8 h at two temperature regimes: in a domestic refrigerator (3–5°C) and at ambient room temperature (20.5–22°C). In the second experiment, eggs were incubated from fertilization to hatching at different experimental temperatures (21, 25, 29, 32 and 35°C) to determine the effects of temperature on the kinetics of white egg appearance, hatching times and hatching quality. Gamete storage at warmer temperatures significantly prolonged viability irrespective of the strain used. In fact, the hatching rate for ova stored at 20.5–22 and 3–5°C for 5 h ranged between 75.2–79.3% and 6.5–9.4% respectively. Loss of viability was most noticeable after 6 h storage at ambient room temperature. Post‐storage viability significantly declined after 2 h exposure to the domestic refrigerator temperature. No hatching of normal larvae took place after 8 h post‐storage time. Results from the second experiment showed that time to maximum whitening of eggs was both strain‐ and temperature‐dependent. The time to maximum mortality of eggs was shorter in the Layo strain (LS) than in the Noun strain (NS), regardless of incubation temperature. The appearance of white eggs was shorter with increasing incubation temperatures. Hatching times decreased with increasing temperature, regardless of strain. Hatching took place from 21 to 27 h and 19 to 24 h after fertilization at temperature of 29°C, respectively, for NS and LS. The length of the hatching period was remarkably shorter for LS than NS at any tested incubation temperature, except 35°C. No hatching took place at 21°C. The highest proportion of normal larvae occurred at 25 and 29°C, respectively, for NS and LS. Hatching rate was highest at 25 and 29°C, respectively, for NS and LS. There was a significantly higher proportion of deformed larvae at 35°C regardless of the strain.     

Studies on cryopreservation of eggs from rainbow trout (Salmo gairdneri) and coho salmon (Oncorhynchus kisutch)

The effect of pre-freezing treatments as well as freezing of inseminated, not water-activated eggs from rainbow trout Salmo gairdneri, and coho salmon, Oncorhynchus kisutch, was investigated in relation to survival and further development.Effects above freezing temperatures included: the temperature at insemination, viability of inseminated and unactivated eggs after storage, suitability of an incubation medium and the tolerance of eggs to various levels of the cryoprotectant dimethylsulfoxide (DMSO). Freezing experiments included: investigating the action of DMSO (0, 1, 2 mole) and the tolerance of coho eggs to temperatures between ?4.6 to ?30°C. Insemination temperatures between 0.5°C and 9.8°C (coho eggs) as well as incubation in an artifical medium (1-0°C) for 80 min (rainbow trout eggs) and 170 min (coho eggs) did not influence subsequent fertility. Storage of inseminated and unactivated rainbow trout eggs for 135 min and beyond reduced egg fertility. DMSO at 2 and 4 mole was detrimental to coho eggs (1-0°C). One mole DMSO had no (coho) or reduced (rainbow trout) influence on egg fertility when it was added gradually.In the presence of 1 mole DMSO most eggs remained unfrozen (67–89%) when kept for 10 min in frozen artificial medium (?4.6%) and 27–32% subsequently reached the eyed stage (control = 100). Further cooling (0.3°C/min) to ?10°C was still tolerated (62% unfrozen, 22% eyed eggs) but not to ?20°C (6% unfrozen, no development) and ?30°C (no survival). Use of 2 mole DMSO did not improve the results.     

Reducing bacterial density in the semen of rainbow trout (Oncorhynchus mykiss) by applying gradient centrifugation and swim‐up washing methods

The objective of this study was to demonstrate the effectiveness of disinfection procedures to reduce bacterial load of rainbow trout, Oncorhynchus mykiss (Walbaum 1792), semen. Fresh semen was obtained from 3–4‐year‐old male species by abdominal sampling of sperm into pre‐cooled test tubes. After sperm cryopreservation and thawing, experiments were accomplished at 4–9°C. ‘Swim‐up’ and gradient centrifugation were used as a sperm washing method with commercial kits. Phosphate buffered saline was also used as washing solution. Bacterial growth tests were employed before and after washing the semen samples. Samples were inoculated on tryptic soy agar (TSA), modified Anacker and Ordal agar (MAOA) as well as brain heart infusion (BHI) agar. After using ‘swim‐up’ method for washing the semen, many bacterial colonies were observed. However, after semen washing with gradient centrifugation, lower bacterial growth was observed on TSA, MAOA and BHI. Some motile (40%) spermatozoa were obtained doing gradient washing procedure. Although sperm motility was not satisfactory, apparently the gradient centrifugation method reduced bacterial contamination as known from the mammalians.     

In vitro storage of fish oocytes: effect of storage temperature,media conditions and storage duration on fertilization and larval hatchability of Indian major carp,rohu (Labeo rohita)

The viability of matured oocytes stored in vitro were assessed using carp ovarian fluid (OF) and artificial carp ovarian fluid (ACOF) under different temperature regimes (4, 24, 26, 28 and 30°C) for different storage durations (0, 60, 120, 150 and 180 min). Significantly higher fertilization (74%) was achieved when the oocytes were stored using ACOF and 65% in OF after 180 min at 28°C. Similarly the hatching rates of larvae were significantly higher in the ACOF and OF, that is, 64% and 47%, respectively, after 180 min of storage. The oocytes kept in the storage containers with ACOF having 65% moisture level showed a significantly higher fertilization rate than the 59% moisture level. This study demonstrated that unfertilized matured oocytes (eggs) of rohu can be stored in vitro for 180 min without compromising the viability (fertilization and hatching) to a great extent in OF and ACOF.     

Effect of Frozen Storage Temperature on Quality-Related Changes in Rainbow Trout (Oncorhynchus mykiss)

The effect of frozen storage temperature on quality-related parameters of rainbow trout (Oncorhynchus mykiss) muscle was studied in the interval from ?10 to ?80°C on samples stored for 1 to 18 months. The following quantities were measured: drip loss, water holding capacity and water distribution, color, lipid oxidation (thiobarbituric acid-reactive substances, TBARS), and membrane stability (enzyme activity). No effect of temperature on drip loss, water holding capacity, water distribution, or membrane stability was observed for samples stored below ?20°C, whereas storage at ?40°C or lower compared to ?30°C or higher resulted in a reduced level of secondary lipid oxidation (TBARS). No advantage was gained by using temperatures below ?40°C for frozen storage of trout regarding any of the properties investigated.     

Influence of storage conditions on viability of quiescent copepod eggs (Acartia tonsa Dana): effects of temperature, salinity and anoxia

Copepods have proven to be an ideal source of live food for the production of marine fish larvae in aquaculture. Therefore, there is a need to develop new methods for production and storage of copepod eggs that can be hatched and used at fish farms. In the present study quiescent eggs of Acartia tonsa were stored for periods up to 35 weeks at different temperatures, salinities and oxygen conditions in a full factorial experiment. None of these storage conditions seemed to induce diapause in eggs even though this has been reported by other authors. The most promising storage conditions were those involving low temperature (<5°C), medium salinity (10–20 ppt) and anoxia. The practical aspects of these results for aquaculture are discussed.     

Egg oxidation status,antioxidant enzyme activities,lipid classes,fatty acid composition profile and embryo survival rates during in vitro oocyte ageing in tench Tinca tinca (Linnaeus, 1758)

The effect of in vitro storage of oocytes on embryos survival rate, egg oxidation status, antioxidant enzyme activities, lipid classes and fatty acid composition profile was investigated in common tench Tinca tinca. In order to identify the role of oxidative stress in the progress of oocyte ageing the levels of thiobarbituric acid reactive substances (TBARS) and carbonyls as indicators of lipid and protein oxidation were measured and the activity of antioxidant enzymes were examined. Stripped ova from six females were stored in cell culture plates at 20°C for up to 10 hr post‐stripping (HPS). The stored ova were fertilized and the embryo survival rates were assessed. The results indicated that tench eggs could be successfully stored in vitro for 4 hr after stripping at 20°C. Superoxide dismutase enzyme activity increased at 6 HPS, whereas glutathione peroxidase and glutathione reductase activities decreased in oocytes during in vitro storage (p < .05) at 4 and 8 HPSs, respectively. The level of malondialdehyde did not show any significant changes during the progress of oocyte ageing. Carbonyls increased up to 2 HPS and thereafter decreased significantly. However, ova ageing did not affect the main lipid class composition and the fatty acid composition of the eggs. Lower quality eggs exhibited lower levels of cholesterol but higher levels of triacylglycerol.     

Semen cryopreservation of salmonid fishes: influence of handling parameters on the postthaw fertilization rate

Using the cryopreservation method of Lahnsteiner, Berger, Weismann & Patzner (1995, Aquaculture Research 26 , 801-807) the influence of allowable variations of methodical parameters (storage of semen before cryopreservation, dilution ratios in the extender, equilibration in the extender, cooling rates, storage of deep-frozen semen in liquid nitrogen, storage of frozen/thawed semen, minimal sperm/egg ratio) was investigated under the aspect of routine utilization. Under optimized experimental conditions, fertilization rates were 90-100% of controls in Oncorhynchus mykiss (Walbaum), Salmo trutta L. f. lacustrisSalmo truttaL.f. fario and Salvelinus fontinalis (Mitchill). The following results were obtained: 1. Storage of untreated semen for more than 1 h before cryopreservation decreased the postthaw fertility. 2. Equilibration of semen up to 20 min in the extender did not affect the postthaw fertility. 3. Optimal dilution ratio of semen in the extender was threefold in Oncorhynchus mykiss and Salvelinus fontinalis. Lower dilution ratios decreased the postthaw fertility, higher dilution ratios did not affect the postthaw fertility. In Salmo trutta f. lacustris and Salmo trutta f. fario, which have a higher sperm density, optimal dilution ratio of semen in the extender was fivefold to sevenfold. 4. In Oncorhynchus mykiss, as in Salmo trutta f lacustris and Salmo trutta f. fario, the optimal freezing height was at 1.5 cm above the level of liquid nitrogen (-110 ± 2^oC); in Salvelinus fontinalis it was 2.5 cm above the level of liquid nitrogen (-92 ± 2^oC). Changes in the freezing height of 0.5 cm (about 10^oC) resulted in a significant decrease of postthaw fertility. 5. Storage of deep-frozen semen for up to 370 days in liquid nitrogen had no influence on its postthaw fertilization rate. 6. Storage of frozen/thawed semen for 30 s before insemination significantly decreased its postthaw fertility. 7. Reliable minimal sperm:egg ratio to obtain fertilization rates of 90-100% of control was 3-5 X 10⁵ spermatozoa egg^-1.     

Egg hatching rate and fatty acid composition of Acartia bilobata (Calanoida,Copepoda) across cold storage durations

To investigate egg storage capacity of the copepod Acartia bilobata for aquaculture interest, we tested hatching success rate (HSR) of inclusive eggs (mixture of all egg types) after 4°C storage. The HSR peaked after 14 days storage when incubating at 28°C for 48 hr (85.8 ± 1.6%) and 72 hr (87.6 ± 0.9%), then gradually declined until 1 year (48 hr: 7 ± 0.6%; 72 hr: 19.4 ± 3.9%). Reallocation of fatty acid profile suggests that docosahexaenoic acid (DHA) is correlated with the HSR of A. bilobata eggs. Additionally, we investigated the HSR of diapausing eggs (unhatched eggs after 72 hr incubation of the inclusive eggs) after 4°C storage. Their HSR peaked after 14 days storage (48 hr:75.3 ± 3.5%; 72 hr:78.2 ± 2.1%), then gradually declined until 60 days (48 hr HSR:42.1 ± 2.3%; 72 hr HSR:53.0 ± 3.2%). Overall, we illustrated the hatchability of diapausing and quiescent eggs of A. bilobata after 4°C storage. The cold storage capacities were low (<60% HSR after 60 days), and it could be limited by the egg DHA content. Our findings provide implications for future studies aiming to improve cold storage techniques of tropical copepod eggs for aquaculture applications.     

Long-term cryopreservation of spermatophore of the giant freshwater prawn, Macrobrachium rosenbergii (de Man)

Long‐term cryopreservation of the giant freshwater prawn, Macrobrachium rosenbergii, spermatophores using glycerol (Gly) and ethylene glycol (EG) as cryoprotective agents (CPAs) was studied. The tolerance of sperm to cryopreservation was evaluated on the basis of sperm survival and fertilizing ability. The survival of the sperm was determined by trypan blue staining, while the fertilizing ability was assessed from artificial insemination of the cryopreserved spermatophores. The rates of embryo survival on day 5 after spawning and of spermatophores capable of producing embryos survived to hatching were determined. Storage of spermatophores at ?20°C without CPA for a short period of up of 1–5 days decreased the sperm survival significantly and did not preserve fertilizing ability. Preservation at ?20°C in the presence of 10% or 20% Gly or of 10% or 20% EG offered a simple and efficient short‐term storage up to 10 days. For a long‐term storage, cryopreservation in the presence of 20% EG at ?196°C was more efficient than at ?20°C. High sperm survival rates and high fertilizing ability were recorded from those cryopreserved at ?196°C for up to 150 days. High sperm survival rates with moderate levels of fertilizing ability were obtained from those cryopreserved at ?20°C for not more than 30 days. The results indicate that preservation at ?196°C with 20% EG is a suitable procedure for long‐term storage of the giant freshwater prawn spermatophores.     

Effects of temperature and dietary n-3 and n-6 fatty acids on endocytic processes in isolated rainbow trout (Oncorhynchus mykiss,Walbaum) hepatocytes

Effects of different incubation temperatures (2, 8, 14 and 20°C) and hepatocyte membrane fatty acid composition on the rate of internalization and lysosomal degradation of the ligand, mannosylated albumin, that is taken up by receptor-mediated endocytosis, were investigated in rainbow trout (Oncorhynchus mykiss, Walbaum). The fish were kept at a water temperature ranging from 9 to 14°C and fed pelleted diets coated with either capelin oil (control), EPA/DHA-concentrate (rich in n-3 polyunsaturated fatty acids) or soybean oil (rich in n-6 unsaturated fatty acids) for at least 3 months prior to sampling. The endocytic uptake mediated by the mannose receptor was very efficient at all temperatures studied. Lysosomal degradation, on the other hand, came to a halt below 8°C. The activation energies for uptake and degradation were 54.6 and 164.2 kJ/mol respectively. No negative effects of increased amounts of either n-3 or n-6 fatty acids were observed on the endocytic parameters studied. On the contrary, multivariate analysis indicated a positive relationship between high levels of n-6 fatty acids and low unsaturation index in the phosphatidylcholine (PC) fraction of the hepatocytes and the internalization rate of 2°C, meaning that the rate of receptor-mediated endocytosis may be affected by membrane fatty acid composition.     

Biopreservative effect of pediocin ACCEL on refrigerated seafood

To investigate the biopreservative effectiveness of pediocin ACCEL on refrigerated seafoods, fresh fish fillets were immersed in various concentrations of pediocin ACCEL and then stored at either 4° or 0°C. Samples treated with nisin were used as a positive control. The aerobic plate counts (APC) of samples with bacteriocins were <2.0 log₁₀cfu/g (log cfu/g) after 2 days storage at 0°C, except that with 1500 IU/mL of pediocin ACCEL. The APC of samples with nisin were >2.0 log cfu/g after 2 days storage, while those with pediocin ACCEL occurred after 1 day storage at 4°C. In refrigerated seafoods, pediocin ACCEL and nisin suppressed the growth of inoculated Listeria monocytogenes during 2- and 1-week storage at 4°C, respectively. Compared with nisin, the pediocin ACCEL was considered to be more effective on the suppression of L. monocytogenes growth in refrigerated seafoods during 2-week storage at 4°C.     

Egg production in the salmon louse [Lepeophtheirus salmonis (Krøyer)] in relation to origin and water temperature

Egg strings of salmon lice, Lepeophtheirus salmonis (Krøyer 1837), collected from farmed and wild Atlantic salmon had similar length and number of eggs string^?1. Egg production was investigated at water temperatures from 7.1 °C to 12.2 °C. A regression model indicated that at low temperatures egg strings were longer and had more eggs. Mean length of single eggs was significantly smaller and the percentage of non‐viable eggs in the strings was higher at 7.1 °C than at 12.2 °C. Adult females survived for up to 191 days at 7.2 °C, and during this period 11 pairs of egg strings were produced.     

Effects of light and short-term temperature elevation on the 48-h hatching success of cold-stored Acartia tonsa Dana eggs

The effects of light and short-term temperature elevation on the 48-h egg hatching success (HS) of cold-stored (2 °C) Acartia tonsa Dana (Copepoda: Calanoida) eggs were examined in the present study. The eggs can be stored for up to 7.5 months and maintain their high hatching rate under optimal conditions. Intensively produced eggs from the copepod A. tonsa may be hatched and used as an inoculum for producing copepod nauplii as live feed for fish larvae. The HS for eggs that were directly exposed to LED light declined rapidly after 1 month of storage (from 91 to 25 %), and these eggs did not hatch at all after 3 months of storage. The highest HS found was for eggs stored in complete darkness. The HS for eggs stored in normoxic (≥7 mg DO L^?1) and anoxic (≤0.03 mg DO L^?1) seawater was not affected by short-term temperature transitions from 2 °C up to 9 or 17 °C for a period of 12 or 24 h, when hatched 1 week post-exposure. The global mean HS for eggs stored in normoxic seawater was 85.9 % and significantly lower compared to eggs stored under anoxic conditions after 3 weeks of storage (91.8 %) (P = 0.001; SNK).     

A study of bacteria present within unfertilized salmon eggs at the time of spawning and their possible relation to early lifestage disease

Abstract. The eggs of 30 female chinook salmon, Oncorhynchus tshawvtscha (Walbaum), were collected at spawning. Some eggs from each fish were collected for bacteriologic study. Two salmon produced eggs judged to be of poor quality which were not used. The remaining 28 of the 30 groups of eggs were fertilized from a single sperm pool and the eggs incubated in separate groups. Mortality data on the developing salmon were recorded regularly through the twelfth week on feed. Unfertilized eggs from each group were surface-disinfected with an iodine solution, then crushed and subjected to a culture procedure designed to permit growth of as many bacterial types as possible. Bacteria were cultured and identified, and a comparison made of the types of organisms present in eggs from groups which later incurred high or low mortalities. Bacteria were recovered from both groups of salmon eggs. Although no single organism could be identified as a cause of increased mortality, the more frequent occurrence in the eggs of the 'high mortality' group of species of Vibrio, Listeria, Corynebacterium and Staphylococcus suggests that these bacteria may play a role. It is suggested that the cause of so-called early lifestage disease of salmon is multifactorial.