Bacterial Colonization of Cod (Gadus morhua L.) and Halibut (Hippoglossus hippoglossus) Eggs in Marine Aquaculture

Aquaculture has brought about increased interest in mass production of marine fish larvae. Problems such as poor egg quality and mass mortality of fish larvae have been prevalent. The intensive incubation techniques that often result in bacterial overgrowth on fish eggs could affect the commensal relationship between the indigenous microflora and opportunistic pathogens and subsequently hamper egg development, hatching, larval health, and ongrowth. Little information about the adherent microflora on fish eggs is available, and the present study was undertaken to describe the microbial ecology during egg development and hatching of two fish species of potential commercial importance in marine aquaculture. Attachment and development of the bacterial flora on cod (Gadus morhua L.) eggs from fertilization until hatching was studied by scanning electron microscopy. The adherent microflora on cod (G. morhua L.) and halibut (Hippoglossus hippoglossus) eggs during incubation was characterized and grouped by cluster analysis. Marked bacterial growth could be demonstrated 2 h after fertilization, and at hatching eggs were heavily overgrown. Members of the genera Pseudomonas, Alteromonas, Aeromonas, and Flavobacterium were found to dominate on the surface of both cod and halibut eggs. The filamentous bacterium Leucothrix mucor was found on eggs from both species. While growth of L. mucor on halibut eggs was sparse, cod eggs with a hairy appearance due to overgrowth by this bacterium close to hatching were frequently observed. Vibrio fischeri could be detected on cod eggs only, and pathogenic vibrios were not detected. Members of the genera Moraxella and Alcaligenes were found only on halibut eggs. Caulobacter and Seliberia spp. were observed attached to eggs dissected from cod ovaries under sterile conditions, indicating the presence of these bacteria in ovaries before spawning. Adherent strains did not demonstrate antibiotic resistance above a normal level. Attempts to regulate the egg microflora by incubation of gnotobiotic eggs with defined antibiotic-producing strains did not result in persistent protection against subsequent colonization by the microflora of the incubator.     

Effects of human chorionic gonadotropin and gonadotropin releasing hormone analogue on plasma steroid hormones and spawning performances in golden rabbitfish Siganus guttatus

In this experiment, golden rabbitfish (Siganus guttatus) were allocated between three treatment groups. The fish were injected with saline, human chorionic gonadotropin (hCG) and D-Ala⁶, Pro⁹-Net-mGnRH. After injection, in 6 hr intervals, blood plasma samples were collected for steroid hormone (testosterone [T] in males and estradiol-17β [E₂] in females) using enzyme immune assay (EIA). In male fish, T levels significantly increased and reached 170 and 650 pg/ml for hCG and D-Ala⁶, Pro⁹-Net-mGnRH treatments, respectively. Then T levels slightly decreased until 24 hr post injection. There were no significant changes of T levels in saline treatment. In female fish, we found significant changes in E₂ levels at 2,567 and 524 pg/ml at 12 hr post injection in hCG and D-Ala⁶, Pro⁹-Net-mGnRH treatments, respectively. No significant differences of E₂ levels were observed in saline group. In the second experiment, we injected 100 golden rabbitfish with both hCG and D-Ala⁶, Pro⁹-Net-mGnRH. Fish spawned successfully when hCG and D-Ala⁶, Pro⁹-Net-mGnRH were given individually and in combination. Latency periods were between 46–64 hr with an average fertilization rate of 70%–90% and hatching rate of 56%–74%. The embryonic duration was 16–20 hr. The saline-injected group produced no spawning. Our findings contribute to further understanding of exogenous hormones impact on golden rabbitfish reproductive endocrinology, refining breeding protocol and implications for fish propagation.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Extension of sperm motility leads to increased rates of fertilization and hatching in curimba,Prochilodus lineatus

The objective of this study was to evaluate the effects of six activating solutions on duration of sperm motility, fertilization rate (FR), and hatching rate (HR) of Prochilodus lineatus (Valenciennes, 1837). The activating solutions (SA) used were: SA₀ (199 mOsm kg^?1, pH 8.5), SA₁ (138 mOsm kg^?1, pH 7.5), SA₂ (256 mOsm kg^?1, pH 7.5), SA₃ (131 mOsm kg^?1, pH 10), NaCl (92 mOsm kg^?1, pH 7.5) and distilled water (32 mOsm kg^?1, pH 7.5). SA₁ induced the highest motility, FR and HR, compared with the other activating solutions. The lowest motility was obtained with SA₀, with no fertilization or hatching, whereas motility was zero with SA₂ and SA₃. It is possible to conclude that the solution SA₁ can be used for the activation of gametes during fertilization in induced reproduction of curimba to achieve higher fertilization and hatching rates. Thus, it was found that the osmolality and pH of activating solutions, probably with the participation of dissolved substances therein, are the main factors acting on semen motility after activation.     

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: Effects of extender supplemented with different antioxidants on sperm motility,velocity and fertility

In present study, it was examined whether addition of different antioxidants to the cryopreservation extenders had an effect on semen post-thaw fertility and motility in rainbow trout (Oncorhynchus mykiss) and also it was investigated the sperm characteristics post-thaw sperm characteristics and fertility. The collected semen was pooled to minimize individual variation. Each pooled ejaculate was split into 12 equal aliquots and diluted with base extenders supplemented with the antioxidants, and a base extender with no additives (control). The pooled semen samples diluted at the ratio of 1:10 by the extenders were subjected to cryopreservation. Antioxidants were separately added to the extenders (one per experimental group): catalase (250 U/l), superoxide dismutase (250 U/l), peroxidase (250 U/l), oxidized glutathione (1.5 mmol/l), reduced glutathione (1.5 mmol/l), l-methionine (1.5 mmol/l), uric acid (0.25 mmol/l), l-ascorbic acid (0.5 mmol/l), α-tocopherol (2.0 mmol/l), β-carotene (0.5 mmol/l) and carnitine (0.5 mmol/l). After dilution the semen was aspirated into 0.25 ml straws, the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Our results indicated that the post-thaw motility rate increased in extenders supplemented with uric acid, l-methionine, SOD, l-carnitine, α-tocopherol and l-reduced glutathione (p < 0.05). The motility duration of frozen thawed semen increased in extenders supplemented with uric acid, l-methionine, SOD, α-tocopherol and l-reduced glutathione (p < 0.05). Fertilization rate and hatching rate of frozen-thawed semen was not affected by the tested antioxidants. Consequently, the tested antioxidants affected the motility parameters and cryopreservation extenders could be supplement with antioxidants. This study suggested usage of antioxidants in the cryopreservation of rainbow trout.     

Induced ovulation and embryonic development of ocellated puffer, Takifugu ocellatus

Acute injections of different hormones to induce ovulation in mature ocellated puffer, Takifugu ocellatus, collected from natural waters during the spawning season, were carried out to develop a reliable protocol for mass production of seed in this species. All experimental fish were divided into seven groups treated with: a saline injection (control), single or two injections of luteinizing hormone‐releasing hormone analog (LHRH‐a; single injection: 50 μg kg^?1, two injections: 10 and 40 μg kg^?1), single or two injections of pituitary (single injection: 6 mg kg^?1, two injections: 1 and 5 mg kg^?1) and single or two injections of human chorionic gonadotropin (hCG; single injection: 2500 IU kg^?1, two injections: 500 and 2000 IU kg^?1), respectively. The percentage of fish that ovulated in six hormonal treatments reached 100%, either with a single injection or with two injections whereas the fish in control group failed to spawn. There were no significant differences among all hormonal treatments in egg production, fertilization rate, or hatch rate (P > 0.05) except time to ovulation between a single injection group and the two‐injection group (P < 0.05). The fertilized eggs of ocellated puffer were spherical, demersal, and adhesive. They had a mean oocyte diameter of 1.487 ± 0.106 mm (range: 1.404–1.560). The egg membrane was transparent and yolk was buff in color, containing a cluster of small oil globules. Thirty‐four successive stages of embryonic development were identified and characterized. Fertilized eggs incubated at 18–20°C generally commenced hatching at 144 h after fertilization. Newly hatched larvae were about 3.26–3.45 mm in length. The induced ovulation technique using acute injections of hormones is an important step in the development of the culture of the ocellated puffer.     

Cryopreservation of sperm from the coral Acropora humilis

Corals are sensitive to minute changes in their environments, and their continued existence is substantially threatened by the increasing number of destructive anthropogenic activities and unprecedented rates of climate change. Although cryopreservation has been successfully to preserve mammalian gametes for decades, coral cryopreservation was attempted for the first time less than 15 years ago, and freezing protocols exist for only a handful of coral species. The present study developed a cryopreservation protocol for the sperm of the common Indo-Pacific reef-builder Acropora humilis. Colonies of reefs of Sattahip Bay, Chonburi Province, Thailand were collected from 3 m depth with a mesh net during a spawning event. Immediately after collection, the sperm were isolated and subjected to a two-step freezing method featuring DMSO, polyethylene glycol, or methanol as the cryoprotectant. Viability and motility were assessed via a bioluminescence technique and a “computer-assisted semen analysis, and it was found that a 15-min equilibration with 2 M DMSO followed by cooling at 41.7 °C was the optimum cryopreservation protocol for A. humilis sperm. The post-thaw sperm achieved 45% fertilization success, and 35% of the fertilized eggs developed into blastopore larvae. The present optimized protocol can therefore facilitate the preservation of sperm for future propagation efforts of this species and provide an experimental platform for optimizing cryopreservation protocols for gametes of other scleractinian coral species.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Assessing the In Situ Fertilization Status of Two Marine Copepod Species,Temora longicornis and Eurytemora herdmani; How Common Are Unfertilized Eggs in Nature?

We utilized an egg staining technique to measure the in situ fertilization success of two marine copepod species, Temora longicornis and Eurytemora herdmani from May to October 2008 in coastal Maine and correlated fertilization success with environmental conditions in their habitat. T. longicornis is a free spawning species that releases eggs into the ambient seawater after mating. In contrast, E. herdmani carries eggs in an egg sac until they hatch. The proportion of fertilized eggs within E. herdmani egg sacs was significantly higher than the freely spawned clutches of T. longicornis. This may be a result of the asymmetrical costs associated with carrying vs. spawning unfertilized eggs. T. longicornis frequently laid both fertilized and unfertilized eggs within their clutch. T. longicornis fertilization was negatively associated with chlorophyll concentration and positively associated with population density in their local habitat. The fertilization status of E. herdmani egg sacs was high throughout the season, but the proportion of ovigerous females was negatively associated with an interaction between predators and the proportion of females in the population. This study emphasizes that, in addition to population level processes, community and ecosystem level processes strongly influence the fertilization success and subsequent productivity of copepods.     

The appearance of beta-1,3-glucanase in hatching embryos of the sea urchin, Echinometra vanbrunti.

Two characteristics of fertilizing sea urchin eggs are the elevation of a fertilization membrane and the excretion of a β-glucanase. Of 13 species tested, one species, Echinometra vanbrunti, lacks both characteristics. No β-glucanase exists in the eggs and cleavage stages. However, β-glucanase appears at hatching and is secreted to the sea water during and after the hatching period. The enzyme may function in the hatching process. The hypothesis is presented that the β-glucanase excreted by eggs of other sea urchin species may function in the elevation of the fertilization membrane.     

The cryopreservation of spermatozoa of the burbot,Lota lota (Gadidae,Teleostei)

The cryopreservation of spermatozoa of a teleost fish, the burbot, Lota lota (Gadidae) was investigated. Cryopreserved semen had the highest motility rate (46.6+/-8.0%, fresh semen control 86.5+/-8.2%) and fertility (78.1+/-2.7% embryo survival in hatching stage, fresh semen control 82.2+/-2.9%) when 10% methanol, 1.5% glucose and 7% hen egg yolk were used as cryoprotectants. Freezing was performed in 0.5-ml straws in the vapour of liquid nitrogen at 1cm above the level of liquid nitrogen and thawing in water at 25 degrees C for 20s. For optimal fertilization cryopreserved semen was first mixed with the eggs and then 25 or 50 mmol/L NaCl solution (pH 8.5) was added at a ratio of 1:24 (semen:saline solution). Under these conditions fertilization ratios in the range of fresh semen control were obtained at minimal sperm to egg ratios of 1.7 x 10(6):1. Fertilization with cryopreserved semen had no influence on the embryonic development, as the ratio of embryos which stopped development and the ratio of embryonic malformations were similar to fresh semen.     

Development of eggs,larvae and juveniles of the puffer,Takifugu chrysops,reared in the laboratory

The artificial fertilization of the puffer,Takifugu chrysops (Hilgendorf), was carried out at Sajima in Yokosuka City on May 22, 1984. Hatched larvae were reared for a period of about 150 days. The spawning period seems to extend from mid to late May in the eastern part of Sagami Bay. The eggs were spherical, pale milky white and semitransparent, demersal and adhesive in nature, measuring 1.32±0.04 mm in diamter, and with a cluster of small oil-globules. The incubation period was about 162 hours at a water temperature of 17.4 to 21.8°C. During embryonic development, the only pigment cells that appeared on the embryo were the black chromatophores. The newly hatched larvae measured from 2.72 to 3.06 mm TL, averaging 2.87±0.1 mm TL, and 22–23 (9 + 13?14) myomeres. At yolk absorption, 4 days after hatching, the larvae attained 3.64–3.79 mm TL. On the 11th day, postlarvae averaged 4.69±0.24 mm TL. Larval finfolds disappeared and rudimental dorsal, anal and caudal fins were formed. There were two large clusters of melanophores, one on the back, exteding from the mid-base of the dorsal fin to the caudal peduncle region, the other along the anal fin base. The color of the body began to turn pale green to brownish-orange and spinelike scales appeared on the belly. Eighteen days after hatching (7.02±0.27 mm TL), the caudal notochord began to turn up and a “constriction” appeared on the posterior margin of the caudal fin membrane. This notch moved upwards as the notochord upturning advances. The larvae attained full fin ray counts and reached the juvenile stage at 9.1-9.5 mm TL, 24 days after hatching. Characteristic black blotches on the back and specific brownish orange body color appeared at the stage of 20 mm TL, 24 days after hatching. The growth during the larval stage and early juvenile stage (24 to 51 days after hatching) were expressed by the following equations, wherey is total length (mm) andx is days after hatching.y ₁=2.8424× 1.0509⁹ (0≦x≦24)y ₂ = 3.7872×1.0372^x (24≦x≦51)     

The possibility of obtaining intergeneric hybrids via White Kołuda (Anser anser L.) goose insemination with fresh and frozen-thawed Canada goose (Branta canadensis L.) gander semen

The objective of the present experiments was to produce the intergeneric hybrids of domesticated and wild goose via artificial insemination with fresh and frozen-thawed semen. The experiments were carried out during two successive goose reproductive seasons, on eight five-year-old Canada Goose (Branta canadensis L.) males used as semen donors and 16 two-year-old White Ko?uda geese designated to fertility tests. Pooled semen was collected twice a week by the dorso-abdominal massage. In freshly collected semen, ejaculate volume, color, consistency, degree of fecal or blood contamination, spermatozoa concentration, motility, and morphology were evaluated. Part of the semen collected in the first year of the experiment (Experiment 1) was used for geese insemination with fresh semen, while the remainder was frozen. In Experiment 2 all samples were subjected exclusively to freezing procedure. Geese were inseminated once a week with fresh semen in a dose of 80 μl or 160 μl, and twice a week with frozen-thawed semen in a dose of 80 μl (160 μl per wk) or 100 μl (200 μl per wk). Eggs were set weekly and incubated up to hatching.The volume of ejaculates varied from 0.100 to 0.470 ml; spermatozoa concentration from 140 to 310 million ml⁻¹; progressive movement was observed in 40 to 60% of spermatozoa; the percentage of total live spermatozoa ranged from 69.3 to 92.0%, the highest percentage (34.0-68.3) was represented by live normal spermatozoa and those with bulb-head (13.3-41.0). Cryopreservation caused a decrease in percentage of motile cells to 30%; total live spermatozoa contribution by 27.2%p, including those live normal by 15.9%p (in relation to the fresh semen), bulb-head spermatozoa by 10.9%p, and increase (by 5.9%p) in number of spermatozoa with other deformations. Goose insemination 1×/week with fresh semen containing about 10.3 million live normal spermatozoa resulted in 66.7% of fertile eggs and with dose higher by 2.8 million spermatozoa (on average) the fertility increased by 20.9%p (up to 87.6% on average). Hatchability from set and fertile eggs was 55.9% and 83.9% vs. 66.3% and 75.6%, respectively. After twice a week insemination with frozen-thawed semen containing about 10.2 million live normal cells 58.2% eggs were fertile; hatchability from set eggs was 42.8% and from fertile eggs 71.7%, while insemination dose increase by 2.7 million spermatozoa per week caused a fertilization increase by 3.8%p (62.0% on average), this increase was not statistically significant, but hatchability from the fertile eggs (95.4%), was significantly (P < 0.05) higher.The use of AI with fresh semen in the creation of intergeneric hybrids of Canada goose males and White Ko?uda females allows a high level of egg fertility to be obtained. Furthermore, one limitation which is the short reproductive season of the Canada goose may be overcome by the use of cryopreserved semen.     

Length rather than year‐round spawning,affects reproductive performance of RAS‐reared F‐generation pikeperch,Sander lucioperca (Linnaeus, 1758) – Insights from practice

The continuous production of large numbers of high quality gametes is essential for aquaculture, particularly in candidate species, such as pikeperch, Sander lucioperca (L.). The common practice of year‐round reproduction is under suspicion of inflicting adverse effects on the quality of the gametes through the disturbance of endogenous rhythms. We hypothesized that such perturbation does not affect RAS‐reared F‐generation broodstock. Reproductive performance (number of eggs) and gamete quality (fertilization and hatching rate) were assessed over the course of 3 years covering six independent, photothermal shifted spawning seasons in a commercial pikeperch hatchery (n = 31 egg batches of F‐generation fish in total). No substantial differences in fertilization or hatching rates could be detected between the individual spawning seasons. Fecundity varied, but there are indications for a size effect on female fecundity with intermediate sized females producing higher number of eggs (~65–70 cm). Low egg quality could be detected in batches of very large fish. In conclusion, size‐specific broodstock composition, but not year‐round reproduction of F‐generation pikeperch spawners affects the reproductive performance.     

Methods for Cryopreservation of Guinea Fowl Sperm

Conservation of indigenous poultry species is an important part of the new Hungarian agricultural strategy. Semen cryopreservation is the most practical method for the long term storage of poultry genetic material. The objective was to compare four protocols for cryopreservation of guinea fowl sperm (slow and fast programmable, freezing in nitrogen vapor, and pellet) and three cryoprotectants (10% ethylene glycol, 6% dimethyl-formamide and 6% dimethyl-acetamide). The efficiency of the methods was examined by in vitro tests (subjective motility scoring, sperm concentration, morphological and live/dead sperm analysis with eosin-aniline staining). Thereafter, the two most promising methods were tested by artificial insemination of frozen-thawed semen (3 times a week for 3 weeks using 300 million spermatozoa/hen), followed by candling of incubated eggs, assessment of fertilization, embryonic death, and hatching rate. The survival rate of live, intact spermatozoa was greatest (p≤0.05) in pellet method and the slow programmable protocol (with 10% ethylene glycol) (28.6 and 23.5%). The two best protocols (based on in vitro assessment of post-thaw semen quality) were subsequently tested in vivo with artificial insemination. The pellet method yielded a 64% fertility rate compared to slow protocol with only 30% fertility. Regardless, both freezing protocols significantly increased embryonic deaths compared to the control group (16,7; 9,1 and 8,3%, respectively). During the 3-week in vivo trial, fertility increased and early embryonic death decreased over time. According to the results the guinea fowl sperm could tolerate the fast freezing in pellet better than the slower freezing rates and resulted acceptable fertility rate.     

Effects of glycerol additions on post-thaw fertility of frozen rainbow trout sperm, with an emphasis on interaction between extender and cryoprotectant

The study was conducted to evaluate the effects of different extenders, cryoprotectants and glycerol additions on the post‐thaw fertility and interactions between extenders and cryoprotectants during cryopreservation. Semen was collected by abdominal stripping from 30 adult male rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and diluted with three different extenders (Erdahl–Graham, Lahnsteiner, glucose‐based) containing 15% DMA, 15% DMSO, 15% DMA + 1% glycerol and 15% DMSO + 1% glycerol at a ratio of 1 : 2. Diluted samples were frozen as 0.1 ml pellets directly on dry ice (solid CO₂, −79°C). Eggs were pooled from 10 females. Fertilization was applied in plastic dishes and 600 eggs were used in each fertilization trial. Pellets were thawed in their own extenders (30°C) at a ratio of 1 : 10. 0.3% NaCl was used for activating motility. Sperm–egg ratio was approximately 3 × 10⁶ sp per egg. Experimental success was determined as the percentage of eyed‐eggs 25 days after fertilization. The highest eyed‐egg rate (49.3%) was obtained from semen frozen with glucose‐based extender containing 15% DMSO + 1% glycerol. Our results indicate that the glucose‐based extender containing DMSO is a useful combination, but that the addition of glycerol does not have a positive effect on post‐thaw fertility, and that interaction of the extender‐cryoprotectant is also important in the cryopreservation of rainbow trout semen.     

Near anoxia and sulfide as possible factors influencing the spatial distribution of Acartia tonsa and Acartia clausi: Comparative evaluation of egg tolerance

In many coastal and estuarine areas the planktonic copepods Acartia tonsa and Acartia clausi show a spatial separation with A. tonsa restricted to brackish waters and confined environments and A. clausi inhabiting areas more influenced by sea water. The hatching and viability of A. tonsa and A. clausi eggs exposed to anoxia and anoxia/sulfide (conditions that are frequent in bottom waters of the most confined areas) was evaluated to determine if these stress factors play a role in the distribution of these species. Subitaneous eggs, spawned by laboratory reared organisms, were incubated in near anoxia (< 7.59 × 10^− 3 mmol O₂ L^− 1) or anoxia/sulfide (∼ 1 mmol L^− 1) for different periods (1, 4, 8 and 15 days), then transferred to normoxic conditions. The exposure of the eggs to near anoxia and sulfide appears to induce the same response (quiescence) in both species. Exposure times ≤ 8 days to near anoxia or anoxia/sulfide did not affect egg viability, while 15 day exposure caused significant declines in hatching success of both species. A significant difference between the effects of near anoxia and anoxia/sulfide was observed when incubation lasted 15 days; hatching of eggs exposed to sulfide being higher than that of eggs exposed to near anoxia for both species. No significant differences were observed between the two species in hatching success of eggs exposed to both near anoxia and anoxia/sulfide (with the exception of eggs incubated in near anoxia for 4 days). The results indicate that the impact of anoxia and sulfide on the eggs of the two Acartia species cannot be a factor explaining the spatial distribution in coastal and brackish environments of these copepods.Feeding experiments on A. clausi were also performed. Suitability of different algal species to rear this copepod was evaluated and the results were compared with data previously obtained for A. tonsa. Differences in feeding needs between A. clausi and A. tonsa are discussed.     

Recent advances in artificial breeding and larval rearing of silver pomfret Pampus argenteus (Euphrasen 1788) in Kuwait

During last several years, Kuwait Institute for Scientific Research (KISR), Kuwait has been trying to develop a sustainable culture technique for silver pomfret (Pampus argenteus). This paper reports the recent research advances in the breeding and rearing of silver pomfret fry at KISR. The eggs collected from wild silver pomfret during spawning seasons of 2012 to 2015 were artificially fertilized under laboratory condition. The average hatching rates of artificially fertilized eggs collected from wild silver pomfrets were 25.6%, 44.8%, 76.7%, and 53.5.0% and average survival rates of metamorphosed fry produced from these eggs were 3.7%, 5.7%, 4.4% and 3.8% for the spawning seasons 2012, 2013, 2014, and 2015, respectively. For captive brood stocks, observation on the spawning time at hourly interval by collecting eggs from the nets set at out-flow of brood tanks showed that the spawning time for captive silver pomfret starts at the time of sunset. In 2012, two groups of captive silver pomfret broods spawned a total of 62x10³ and 66x10³ eggs, but the eggs were unfertilized. No captive spawning occurred during 2013 and 2014 spawning season. In 2015, captive silver pomfret broods in three tanks spawned about 653x10³, 673x10³ and 270x10³ eggs, and in 2016, the broods in four tanks spawned about 669x10³, 22x10³, 3x10³ and 366x10³ eggs. However, from these eggs only 1,400 and 1,000 fertilized eggs were collected which produced 300 and 123 hatched larvae in July 2015 and June 2016, respectively. The larvae produced from the 2015 broods did not survive beyond 56 days of metamorphosed stage while larvae produced from 2016 captive broods survived for 9 days only. The mortality of the larvae from captive brood could be related to the poor egg or milt quality. However, efforts are continued to improve the eggs and sperm quality through proper brood management.     

Egg hatching of two locusts,Schistocerca gregaria and Locusta migratoria,in response to light and temperature cycles

The present study showed that the eggs of the desert locust, Schistocerca gregaria, and the migratory locust, Locusta migratoria, responded to photoperiod by hatching when placed on sand in the laboratory. S. gregaria mainly hatched during the dark phase and L. migratoria during the light phase. The importance of light as a hatching cue depended on the magnitude of the temperature change during the thermoperiod; photoperiod played a more important role in the control of hatching time in both species when the magnitude of the temperature change was small. In addition, the eggs of the two species that were covered with sand did not respond to photoperiod and hatched during both the light and dark phases, indicating that light did not penetrate through the sand. Because locust eggs are normally laid as egg pods and a foam plug is deposited between the egg mass and the ground surface, we tested a possibility that naturally deposited eggs perceived light through the foam plug. The eggs that were deposited and left undisturbed in the sand hatched during the light and dark phases at similar frequencies. These results suggest that the eggs of both locust species responded to light and controlled their hatching timing accordingly but would not use light as a hatching cue in the field. The evolutionary significance of the ability of eggs to respond to light in these locusts was discussed.     

Experimental Parameterisation of Principal Physics in Buoyancy Variations of Marine Teleost Eggs

It is generally accepted that the high buoyancy of pelagic marine eggs is due to substantial influx of water across the cell membrane just before ovulation. Here we further develop the theoretical basis by applying laboratory observations of the various components of the fertilized egg in first-principle equations for egg specific gravity (ρ_egg) followed by statistical validation. We selected Atlantic cod as a model animal due to the affluent amount of literature on this species, but also undertook additional dedicated experimental works. We found that specific gravity of yolk plus embryo is central in influencing ρ_egg and thereby the buoyancy. However, our established framework documents the effect on ρ_egg of the initial deposition of the heavy chorion material in the gonad prior to spawning. Thereafter, we describe the temporal changes in ρ_egg during incubation: Generally, the eggs showed a slight rise in ρ_egg from fertilization to mid-gastrulation followed by a gradual decrease until full development of main embryonic organs just before hatching. Ontogenetic changes in ρ_egg were significantly associated with volume and mass changes of yolk plus embryo. The initial ρ_egg at fertilization appeared significantly influenced by the chorion volume fraction which is determined by the combination of the final chorion volume of the oocyte and of the degree of swelling (hydrolyzation) prior to spawning. The outlined principles and algorithms are universal in nature and should therefore be applicable to fish eggs in general.