Dietary effects on egg production,egg‐hatching rate and female life span of the tropical calanoid copepod Acartia bilobata

Copepods are crucial source of live feeds in the aquaculture industry. In particular, several species of the genus Acartia are considered optimal prey for fish larvae. The species Acartia bilobata has excellent potential for marine larvae culture, as it is easy for mass culture. This study investigated the effects of various algal diets on the egg production and egg‐hatching rate of A. bilobata. The results indicated that the single‐species diet Isochrysis galbana was the most supportive diet for A. bilobata egg production and female life span in all treatments (egg production: 23.85 ± 0.70 eggs female^?1 day^?1 and female life span: 18.00 ± 1.45 days). Nannochloropsis oculata and Tetraselmis chui treatments gave markedly lower egg production and female life span as both single‐species and multiple‐species diets. For the egg hatching‐rate experiment, except for the T. chui treatment, which yielded a considerably lower hatching rate than the other diets, the hatching rate was only slightly affected by the algal diets. These results confirm that A. bilobata, a tropical brackish‐water copepod species, develops rapidly at 28°C and can produce a large number of eggs; therefore, it has considerable potential for larvae culture.     

The effects of temperature and photoperiod on egg hatching success,egg production and population growth of the calanoid copepod,Acartia grani (Calanoida: Acartiidae)

Calanoid copepods, including species of the genus Acartia, are commonly used as larval diets for marine finfish. This study aimed to determine the separate effects of water temperature (18, 22, 24, 28° ± 0.5°C) and photoperiod (24L:0D; 18L:6D; 12L:12D; 8L:18D; 0L:24D) on Acartia grani egg production (EP), hatching rate (EHR) and population growth. Egg production rate was not affected by the two abiotic parameters. A. grani eggs incubated at T24°C and T28°C were the first to achieve 50% hatching rate (23–25 hr), with significant differences at the end of the experiment (48 hr) between T28°C treatment (EHR 88 ± 5%) and T18°C treatment (EHR 65 ± 2%). However, different temperature regimes did not affect final number of individuals in population growth experiment. Still, when eggs were excluded from data, population at lower temperatures (18°C) was mainly composed by the nauplii stage (72%), while at higher temperatures (24°C and 28°C) more than 60% of the population was composed by copepodites and adults. A. grani subjected to long‐day photoperiods had significantly lower EHR (16.7% at 24L:0D; 20.8% at 18L:6D) than at short‐day photoperiods (52.6% at 6L:18D; 50.0% at 0L:24D). In population growth experiment, eggs were the most common life stage after 12‐day culture. Lowest population number was found at constant light conditions (665.0 ± 197.1), suggesting higher metabolic rates and depletion of energy reserves in long‐day conditions. This study expanded knowledge on the biological response of A. grani to separate temperature and photoperiod regimes, and provided ground to improve the culture of this potential life feed species for hatcheries.     

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.     

Effects of commonly used disinfectants on bacterial load,hatchability and survival of Bluefin Sea bream (Sparidentex hasta) eggs

The efficacy of four chemical reagents, iodophor, formalin, hydrogen peroxide and bronopol as fish egg surface disinfectants were evaluated in bluefin sea bream (Sparidentex hasta). Fertilized eggs were counted and subjected to a static bath dip treatment in different concentrations of the above chemicals for 4 min before being incubated at 20 ± 0.5°C for 40 h. Treatment efficacy of the different disinfectants was evaluated by assessing the bactericidal activity, egg hatch percentage and survival of larvae up to 3 days post hatch. Results showed that iodophor at medium concentrations (75 and 100 ppm) was the best of all tested disinfectants in bacterial killing ability (12% reduction in the bacterial counts), egg hatching per cent (99.8% and 99.6% respectively) and larval survival up to 3 days post hatch (50.8% and 54.8% respectively). Formalin was the second best disinfectant at levels of 100 and 150 ppm. Hydrogen peroxide gave good results compared with the control while, bronopol showed discouraging results. In conclusion, iodophor appeared to be suitable for bluefin sea bream eggs disinfection with a 4 min exposure to 75–100 ppm when applied 14–16 h after egg fertilization.     

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).     

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.     

Effect of temperature increase on the embryonic development of Patagonian red octopus Enteroctopus megalocyathus in controlled culture

One of the major problems involved in the controlled cultivation of Patagonian red octopus (Enteroctopus megalocyathus) is its long embryonic period ranging between 150–176 days, after which the hatching of planktonic paralarvae is achieved. The effect of temperature on the incubation of E. megalocyathus eggs was studied with the aim of establishing if a temperature higher than 12°C is effective to accelerate the embryonic development without altering their morphological and physiological conditions. Fertilized eggs obtained under controlled conditions at 11°C ± 0.1 were randomly distributed in 12 water baths of 30 L at 4 temperatures: 12, 14, 15 and 16°C ± 0.1°C. The experiment lasted until egg hatching occurred.The embryo growth rate was accelerated at 15–16°C, so the time spent in embryonic development can be reduced in 15% when compared with embryo development obtained from eggs incubated at 12°C. The embryos showed no significant differences in the final survival and were morphometrically similar in all stages of development at all temperatures. The increase in temperature from 12 to 16°C, even if it allowed a better growth, had high metabolic costs for embryos of E. megalocyathus. The activities of lipases and proteases were affected by interaction between temperature and the embryo stage, with high lipase activity observed in embryos of stage XV incubated at high temperatures and the highest levels of trypsin and chymotrypsin in stage XX at 14°C. The results suggest that 15°C could be the limit temperature to increase growth.     

Dietary fatty acid enrichment during the spawning season increases egg viability and quality in Hippocampus hippocampus

Control of the reproductive cycle is essential to achieve a successful aquaculture industry. Seahorse reared in captivity often shows eggs of poor quality and low offspring survival due to unsuitable rearing and nutritional protocols. This study aimed to evaluate the effect of dietary enrichment, added exclusively during the spawning season, on the egg quality of Hippocampus hippocampus. The effect of enriching mysis throughout the spawning season was analysed in terms of female fecundity, egg viability, egg size and fatty acid profile. The results showed that fecundity was not affected by the enrichment of the diet, but egg viability was increased when females were fed the enriched mysis (84.9 ± 4.1% compared to 62.2 ± 9.4%). Moreover, the content of several PUFA (20:4n‐6, 18:2n‐6, 22:5n‐3, 22:5n‐6, sum of n‐6) decreased throughout the spawning season, especially in the last clutch, in eggs originated from females fed non‐enriched mysis. Eggs from females fed the enriched diet maintained the fatty acid profile throughout the spawning season. In conclusion, enrichment of the diet of H. hippocampus improved egg viability and avoided a decrease in the quality of eggs by maintaining PUFA content in the last clutches of the spawning season.     

Fertilization rate,motility, lipid peroxidation and pH changes after chilled storage of rainbow trout (Oncorhynchus mykiss) eggs and spermatozoa by a RMPI medium

The aim of this study was to evaluate the effects of Roswell Park Memorial Institute (RPMI) 1,640 medium on chilled storage of eggs and spermatozoa of rainbow trout (Oncorhynchus mykiss). After 3 days of storage, eggs in RPMI 1,640 media (pH 8.2, 9 and 10), Cortland medium and coelomic fluid were inseminated with fresh spermatozoa (Experiment 1). Eggs in RPMI 1,640 medium at pH 8.2 shown the lowest thiobarbituric acid‐reactive substances (TBARS, 0.053 ± 0.003 nmol/ml) and pH changes (from 8.20 ± 0.01 to 8.18 ± 0.01), the highest fertilization rate (82 ± 3%). Undiluted and diluted spermatozoa at ratios of 1:2 and 1:9 with RPMI 1,640 media (pH 8.2, 9 and 10) and Cortland medium were inseminated with fresh eggs (Experiment 2). Spermatozoa in RPMI 1,640 medium at pH 9 (1:9) caused the lowest TBARS content (0.037 ± 0.002 nmol/ml) and pH changes (from 9.00 ± 0.01 to 8.98 ± 0.01), the highest fertilization rate (77 ± 2%) and motility parameters. Based on Experiments 1 and 2, eggs and spermatozoa were stored for another 3 days in RPMI 1,640 medium at pH 8.2 and 9 (1:9) respectively (Experiment 3). Fertilization rate of storage eggs and spermatozoa in Experiment 3 was 79 ± 5%, showing successfully storage of rainbow trout gametes with the same medium.     

Reproductive characteristics and variations in the biochemical composition of Neptunea arthritica cumingii crosse through embryonic development

The copulation, egg laying, embryonic development and changes in amino acids and fatty acids in Neptunea arthritica cumingii during embryogenesis were studied to understand the embryo development process and nutritional requirements in the early life phase. The results showed that N. arthritica cumingii has direct development within the egg capsule and the development of embryos was classified into five stages: cleavage, egg swallowing, protoconch forming, shell development and juvenile. Embryos develop through the provision of nurse eggs as an extra‐embryonic source of nutrition. As development continued, the body of the embryo began to coil. After about 70–80 days, young N.arthritica cumingii started to emerge through a hole underneath the capsule. Biochemical results showed that the total amount of amino acids showed a decreasing trend as embryonic development progressed. The content of all nine essential amino acids decreased significantly from the egg‐swallowing stage to the post‐larva stage (p < .05). Concentrations of five of the seven nonessential amino acids also showed a decreasing trend from the egg‐swallowing stage to the post‐larva stage; the exceptions were Ala and Gly. Gly is the only amino acid that consistently increased in concentration during the development process. Most fatty acids increased after the eggs hatched, except for C20:1, C20:2, C22:5 and C22:6 (docosahexaenoic acid, DHA). The data in this study may provide a starting point for the formulation of well‐balanced early‐stage larval diets, although N.arthritica cumingii is still in the exploration stage.     

Year‐round natural spawning,early development,and the effects of temperature,salinity and prey density on captive ornate goby Istigobius ornatus (Rüppell, 1830) larval survival

Natural spawning, early development and larviculture of the ornate goby Istigobius ornatus in captivity were studied for the first time. I. ornatus spawned 46 times from 31 October 2013 to 31 October 2014. Fecundity ranged from 246 to 10,214 eggs per clutch, with an average hatching rate of 77.8% ± 9.9% (M ± SEM). Fertilized eggs (1.31–1.54 × 0.46–0.50 mm in diameter) were adhesive demersal and oval‐shaped. Embryonic development lasted 84 hr at 27.5 ± 0.5°C. Newly hatched larvae [2.12 ± 0.04 mm in total length (TL)] transformed to the juvenile stage completely when TL was 7.79 mm. Effects of different water temperatures (24, 28 and 32°C) and salinities (10, 15, 20, 25, 30, 35 and 40 g/L) on per cent survivals (%) and survival activity indices (SAIs) were tested. Survival was not significantly different under different temperatures; SAIs was significantly higher at 28°C. Larvae showed the significantly higher survival and SAIs at salinities 10–30 g/L than at 35 and 40 g/L. Effect of different prey densities on survival was significantly higher in 7 days post hatch larvae fed 20 and 30 rotifers/ml. These findings could guide future programs in captive breeding technology development and commercial production of other marine ornamental gobies.     

The effects of salinity on reproductive development and egg and larvae survival in the spotted scat Scatophagus argus under controlled conditions

In the present study, we report the first successful instance of controlled reproduction in Scatophagus argus, which has recently emerged as a new aquaculture resource. The controlled reproduction process for S. argus was optimized with regard to salinity acclimation. Gonadal maturation was affected by salinity in both sexes. Levels of plasma 17β‐estradiol (E₂) and 11‐ketotestosterone (11‐kT) were salinity dependent and increased significantly with the duration of acclimation. Plasma levels of gonadal steroids were higher in fish held at 25‰ salinity. The highest gonadosomatic indices (GSI), 15.1 ± 1.6 in the female and 6.4 ± 1.2 in the male, were also observed at 25‰ salinity. Nevertheless, the optimal salinity for S. argus embryonic development and larval culture was 15‰. Thus, the salinity requirement for gonadal maturation and early development are quite different. The use of advanced reproductive technologies combining salinity acclimation and stimulation of luteinizing hormone‐releasing hormone analog (LHRH‐A₂) resulted in a fertilization rate of 83.2%–91.3% and embryonic survival rates of over 90%. Embryos of S. argus at the 2‐cell, blastula, gastrula and pharyngula stages were observed. Most embryos hatched after 21.0 hr of incubation at 28.0 ± 1.0°C. The development of larvae into juveniles was completed at 40–45 days posthatch (dph). In this study, we provide information about the controlled reproduction of S. argus and identify the optimal environmental parameters for S. argus embryonic and larval culture, with the aim of developing reliable reproductive techniques for its mass production.     

Long‐term low‐salinity stress affects growth,survival and osmotic related gamma‐aminobutyric acid regulation in the swimming crab Portunus trituberculatus

A 30‐day experiment was conducted to investigate the effects of long‐term low‐salinity stress on the growth performance and osmotic related chlorine ion channel ligand regulation: gamma‐aminobutyric acid (GABA) content and GABA_A receptor‐associated protein (GABARAP) expression in Portunus trituberculatus. The salinity levels of both the control group and the experimental group were 30 and 12 psu respectively. After rearing for 30 days, the specific growth rate and survival rate were compared between the two groups, and salinity 6 psu was used to test the salinity tolerance. The results were as follows: (a) Both the specific growth rate and survival rate were significant lower in the experimental group (p < 0.05) after 30 days; (b) After challenge with salinity 6 psu for 72 hr, the crabs of experimental group had a 100% survival rate, whereas the crabs of the control group were all dead within 48 hr; (c) The content of GABA and the gene expression level of GABARAP in experimental group were significant different from control group (p < 0.05) after challenge via salinity 6 psu. In the control group, the GABA content increased rapidly from 9.96 ± 2.09 to 42.00 ± 5.94 µg/g; however, in the experimental group, it only increased to 27.82 ± 2.55 µg/g; the gene expression of GABARAP in the experimental group increased to the maximum at 24 hr, then decreased and stabilized at 48 hr, suggesting that GABA and GABARAP were trigged during the early stage of low‐salinity stress resistance.     

A fructose‐based extender protects Colossoma macropomum spermatozoa against chilling injuries

Our study assessed the efficiency of a formulated new extender in maintaining viability and morphological integrity of Colossoma macropomum spermatozoa under chilling storage. Semen was diluted in the test extender and BTS? (Beltsville Thawing Solution) and exposed to a short‐term storage at 4.6 ± 0.6°C for 96 hr. Both extenders were able to maintain 17% ± 8% motile spermatozoa by the end of experiment. Sperm dilution in test extender did not affect the morphologically normal cells (61% ± 6%) up to 48 hr of chilling, being higher than in BTS? (50% ± 6%) (p < 0.05). After 96 hr, samples kept in the test extender had 50% of normal spermatozoa, whereas those kept in BTS? presented only 38% of normal cells. Chilling storage increased the incidence of cells with strongly coiled flagella in BTS^?. Our study is the first to evaluate in detail the spermatozoa morphology as indicative of C. macropomum semen viability. The new extender was able to protect the spermatozoa against increase in coiled flagellum injuries.     

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.     

Effect of thermal stress on Hsp70 gene expression and female reproductive performance of giant freshwater prawn,Macrobrachium rosenbergii

Using Hsp70 as a biomarker, thermal stress impinges on reproductive organs, ovary and hepatopancreas were being analyzed by determining the expression of Hsp70 mRNA inside the organs after the adult inter‐molt females were subjected to thermal treatment at 35, 30 and 28°C (Control). Results showed the expression of Hsp70 mRNA under thermal treatment of 35°C after 2 hr recovery in ovary were upregulated at 2, 4, 6, 12, 24 hr and 30 days compared to control whereas in hepatopancreas under similar treatment, the expression of Hsp70 mRNA were significantly higher than control at 6, 24 hr and 30 days. Frequency of reproductive molt at 35°C showed the ovary of females were failed to develop and only entered common molt along three consecutive molt cycles. For 30°C thermal treatment, the expression of Hsp70 mRNA was significantly higher than control after 2 hr recovery but returned to normal afterwards until 30 days’ thermal treatment. Maternal heat shock for 2 hr at 35°C were found to give significantly lower frequency of reproductive molt and longer duration of ovarian development and incubation period whereas maternal heat shock for 2 hr at 30°C gave lower frequency of reproductive molt, slower development of embryo and lower hatching success compared to untreated control. This study suggests that short and long‐term thermal stress at 30 and 35°C were found to affect the induction of Hsp70 mRNA in reproductive organs of Macrobrachium rosenbergii and also influence their reproductive performance.     

Diphasic sedimentation technique complemented by a statistical approach as a useful tool to the quantification and the determination of dynamics of eggs produced by adult monogenean parasitizing groupers,Epinephelus marginatus (Lowe, 1834), in captivity

Monogenean parasites create problems in confined fish, such as at aquaculture and aquariums facilities. Strategic timing of antihelminthic treatments should be based on the dynamics of monogenean egg production which requires egg quantification, but there is no standardized analytical method. The WHO diphasic sedimentation method for helminth quantification of eggs was applied in samples with tetrahedral and fusiform monogenean eggs from six specimens of confined groupers (Epinephelus marginatus) (31 m³ sea water tank; water temperature 21.1–14.4°C; photoperiod 9:53–15 h light; November 2011 to June 2012). Helminth eggs remain in the pellet, while most organic matter was removed forming a cap. In contrast, monogenean eggs are distributed within both the pellet and cap. The diphasic sedimentation method is suitable to quantify tetrahedral and total egg number by counting those eggs in the pellet and incorporing water temperature data to obtain a statistically significant model expression (R² = 0.943; R² = 0.809), while this approach only weakly predicted fusiform egg numbers (R² = 0.297). Fusiform egg numbers declined along November–December, and increased in February–March, until June. Tetrahedral eggs decreased in November–December, but did not increase until March. The trend of egg populations and best time for antihelminthic treatment application can be set by WHO technique with the statistical approach proposed.     

Effect of incubation temperature on the embryonic development and yolk‐sac larvae of the Pacific red snapper Lutjanus peru (Nichols & Murphy, 1922)

The effect of incubation temperature on embryonic development and yolk‐sac larva of the Pacific red snapper Lutjanus peru were evaluated by testing the effect of 26, 28 and 30°C, as this is the natural thermal interval reported during the spawning season of Pacific red snapper in the Gulf of California, Mexico. Sixteen developmental stages were observed. The incubation temperature affected the rate of development and time to hatching, being shorter at 30 than at 26°C, but no significant effect (P < 0.05) on larval length at hatching was registered. The depletion rate of yolk sac and oil globule was affected by incubation temperature particularly during the first 12 h post hatching (hph). At the end of the experiment (48 hph), significantly (P < 0.05) larger larvae were recorded at 26°C (TL = 3.22 ± 0.01 mm) than at 28° (TL = 3.01 ± 0.02 mm) and 30°C (TL = 2.97 ± 0.05 mm). Incubation of newly fertilized eggs at 26°C produces larger larvae, which may help to improve feeding efficiency and survival during first feeding.     

Strain-specific vital rates in four Acartia tonsa cultures, I: Strain origin, genetic differentiation and egg survivorship

Successful development of planktonic copepods for use as live feed in marine aquaculture relies on optimization of environmental conditions for population growth and egg storage. This study examined strain-specific differences in egg survivorship during cold-storage-induced quiescence in four cultures of the widespread marine calanoid Acartia tonsa Dana, 1849. Experimental cultures were obtained from Øresund, Denmark (DIFRES), Kiel, Germany (KIEL), Turkey Point, Florida, USA (FL), and Mobile Bay, Alabama, USA (AL), and were shown to derive from three highly distinct mitochondrial clades. Eggs from Gulf of Mexico strains had low tolerance for cold storage, and showed very low hatching success after 10 days. In contrast, Baltic Sea strains produced eggs able to tolerate up to 150 days of cold storage, with the DIFRES strain showing the highest egg survivorship during the experiment. Eggs from the Kiel strain showed an increase in hatching over time, indicating the presence of dormant eggs. Parental rearing temperature was also found to affect egg survivorship during quiescence in the DIFRES strain, with lower hatching success observed among eggs produced at 25 °C than at 17 °C. The DIFRES strain is recommended as the optimal strain for use in aquaculture, and conditions for cold storage of eggs are discussed.