Embryonic development and morphology of eggs and newly hatched larvae of Pacific herring <Emphasis Type="Italic">Clupea pallasii</Emphasis>

The embryonic development and morphology of eggs and newly hatched larvae of the Pacific herring Clupea pallasii were described using laboratory-reared specimens originating from the Miyako Bay stock. The eggs were almost spherical in shape, 1.33–1.46 mm (mean: 1.38 mm) in diameter, and had a thick adherent chorion. They had a segmented pale yellow yolk, no oil globule, and a relatively wide perivitelline space. A subgerminal cavity was observed during the gastrula period, whereas the blastocoel did not appear. Mass hatching occurred by 271 h 45 min after fertilization, and the newly hatched larvae were 7.1–7.7 mm (mean: 7.5 mm) in total length with 53–56 myomeres at 9.6°C. The embryonic development of Pacific herring was substantially similar to that of zebrafish Danio rerio, American shad Alosa sapidissima, as well as Atlantic herring Clupea harengus, and generally followed the basic developmental pattern of teleosts. However, Pacific herring larvae hatched at a more developed stage than some other clupeoids, such as Japanese sardine Sardinops melanostictus, and the progressed developmental stage at hatching could be interpreted as an advanced adaptation.     

Morphological, physiological and biochemical parameters characterizing the over-ripening of rainbow trout eggs

In rainbow trout (Oncorhynchus mykiss) freshly ovulated eggs and over-ripened eggs which had been retained in the coelomic cavity for 7, 14 and 21 days were investigated in aspects of morphology, physiology and biochemistry. Egg viability was significantly reduced from 85.9±16.4% in freshly ovulated eggs to 25.1±21.9% in over-ripened eggs which had been retained in the coelomic cavity for 21 days. Further during over-ripening in the ovarian fluid the pH significantly decreased, while the levels of proteins, of esterified and non esterified fatty acids and the activities of aspartate aminotransferase and acid phosphatase significantly increased. Also egg parameters changed: the wet weight of the unhardened eggs increased, the weight increase during hardening and the levels of esterified and of non esterified fatty acids significantly decreased. In freshly ovulated eggs the yolk consisted of a homogenous mass and the perivitelline space was small, but in over-ripened eggs the yolk was non homogenous with numerous vesicular inclusions and the perivitelline space was enlarged. When freshly ovulated eggs were incubated in water the cortical reaction was detectable within 5 min, in over-ripened eggs hardly no extrusion of cortical vesicles was visible and the width of the perivitelline space was very irregular.For the investigated freshly ovulated and over-ripened samples the egg viability significantly correlated with ovarian fluid parameters (pH, protein, non esterified fatty acids, esterified fatty acids, aspartate aminotransferase, acid phosphatase) and egg parameters (weight increase during hardening, weight of the hardened eggs).     

Carbohydrate Metabolism of Vitellogenic Follicles and Eggs of Serranus cabrilla (Serranidae) and Mullus barbatus (Mullidae) and of Embryos of Sparus aurata (Sparidae)

The present study compared the carbohydrate metabolism of pelagic eggs of three marine species, Serranus cabrilla (Serranidae), Mullus barbatus (Mullidae) and Sparus aurata (Sparidae). In particular, in Serranus cabrilla and Mullus barbatus the changes in carbohydrate metabolism from the vitellogenic follicle stage to the ovulated egg stage (final maturation of the egg) were investigated, and in Sparus aurata the changes from the unfertilized egg stage to the late-embryo stage just before hatching were observed. In mature, unfertilized eggs of these three species the qualitative and quantitative composition of carbohydrates, enzymes and metabolites were similar, indicating no differences in carbohydrate metabolism. Glycolysis, gluconeogenesis and the pentose phosphate pathway occurred in the follicles and eggs. From the vitellogenic follicle stage to the mature egg stage glycolytic activity was constant in S. cabrilla and M. barbatus, while the activity of the pentose phosphate pathway and of gluconeogenesis decreased. Quantitative carbohydrate composition changed as the levels of hexose, ketose, 6-deoxyhexose, uronic acid and fructose-6-phosphate increased from the vitellogenic follicle stage to the mature egg stage, while the levels of D-glycerate-2-phosphate and phosphoenolpyruvate decreased. In S. aurata, the investigated parameters of carbohydrate metabolism were similar between unfertilized eggs and eggs in the first cleavage stage. From the first cleavage stage to the late embryo stage the levels of hexose, 2-deoxyhexose, ketose, ribose, uronic acid and phosphoenolpyruvate increased, while the levels of D-glycerate-2-phosphate decreased.     

Induced Ovulation of Southern Flounder Paralichthys lethostigma Using Gonadotropin Releasing Hormone Analogue Implants

Implanted pellets that provide a sustained release of [D-Ala⁶ Des-Gly¹⁰] LHRH-ethylamide (GnRHa) were used to induce maturation and ovulation of Southern flounder Paralichthys lethostigma . Of the 12 females whose ovaries contained follicles with a maximum diameter ≥500 μm, 11 ovulated for the first time within 90 h of hormone implantation. Only 1 fish with a maximum follicle diameter less than 500 μm ovulated within 2 wk after implantation. Ovulated eggs were manually stripped from the females and mixed with sperm from several males. Most females were spawned 1 to 3 times on consecutive days with variable fertility. One female was spawned 11 times producing 668,000 eggs. Fertility was evaluated by examining the incubated eggs for early stages of embryonic cleavage. The percentage of fertile eggs in subsamples of incubated eggs ranged from 7–95%. The results indicate that GnRHa implants can be used to induce repeated ovulation in this species. The variability in fertility is discussed in relation to egg quality.     

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

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

Hormone Induced Spawning of Summer Flounder Paralichthys dentatus

During their first year in captivity, summer flounder Paralicthys denratus were induced to spawn with gonadotropin releasing hormone analogue (GnRHa) implants, injected carp pituitary extract (CPE) or human chorionic gonadotropin (hCG) injections. The percentage of fertile eggs was greatest (69%) in CPE-treated females. CPE, but not GnRHa or hCG, was capable of stimulating oocyte growth (increased follicle diameter during vitellogenesis) followed by ovulation. Fish with maximum ovarian follicle diameters between 180 and 435 μm at the initiation of CPE injections produced the greatest percentage of fertile eggs. For most females, fertilization rate was greatest for the first batch of eggs ovulated. The mean fertilization rate for the first spawn of CPE-treated fish was 42% compared with 14% for the second spawn from the same fish. Fish with maximum initial follicle diameters of 585 40 μm that were implanted with GnRHa ovulated the greatest number of eggs, but fertility was low and variable. Approximately 35% of females injected with hCG ovulated a limited number of eggs, but only one hCG-treated female produced fertile eggs. Only a limited number of spermiating males were available for spawning trials. Hormone treatments used on females were ineffective for inducing or maintaining spermiation in male summer flounder.     

Thyroid hormones in eggs of various freshwater,marine and diadromous teleosts and their changes during egg development

Thyroid hormone concentrations in unfertilized eggs of 26 species of various freshwater, marine and diadromous teleosts were examined, together with changes in their concentrations during egg development in some species. Significant quantities of both thyroxine (T₄) and triiodothyronine (T₃) were found in eggs of all species examined. Mean T₄ and T₃ concentrations in eggs varied from 0.04 (marbled sole) to 15.00 ng/g (chum salmon), and from 0.07 (goldfish) to 9.95 ng/g (Pacific herring), respectively. T₄ concentrations were significantly greater than T₃ concentrations in eggs of most freshwater fishes, whereas T₃ concentrations were greater in seawater fishes. During the course of development, thyroid hormones in eggs decreased markedly before hatching. These findings suggest that thyroid hormones are consistently present in teleost eggs, and thus may play an important role in the egg development.     

Assessing the abundance and distribution of eggs of sardine, Sardinops sagax, and round herring, Etrumeus whiteheadi, on the western Agulhas Bank, South Africa, using a continuous, underway fish egg sampler

A continuous, underway fish egg sampler (CUFES) was employed to assess the abundance and distribution of eggs of both sardine, Sardinops sagax , and round herring, Etrumeus whiteheadi , on the Western Agulhas Bank, South Africa, during September 1996. Samples were collected while underway along six inshore/offshore transects, and at stations along the transects. Volumetric estimates of egg density (eggs m⁻³) from on-station CUFES samples were highly correlated with both volumetric and areal (eggs m⁻²) estimates of egg density from samples collected from CalVET net hauls at these stations, demonstrating the validity of this novel sampling technique. Sardine and round herring eggs were encountered in a band running parallel to the coast and extending from 10 to 30 nautical miles offshore to the shelf edge, and highest egg densities were associated with strong north-west-flowing currents in the region of the shelf edge. Collecting samples while underway increased the precision of the estimate of mean egg density for sardine eggs but not for round herring eggs. The use of CUFES in obtaining a fine-scale resolution of sardine egg distribution, and as a tool for stock assessment, are discussed.     

Factors driving spatial variation in egg survival of an ecologically and culturally important forage fish

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Egg quality in rainbow trout: The relation between egg viability,selected aspects of egg composition,and time of stripping

Batches of eggs (1 batch/female) from 17 rainbow trout in their first spawning season were stripped and reared separately, and the percentage which hatched and the percentage which reached the stage of first feeding were determined. The fertilised egg batches were analysed for egg wet weight, egg dry weight, chorion weight and levels in the eggs of free, bound and total lipid, precipitable protein, protein phosphorus, lipid phosphorus, calcium and iron. All determinations were made on single eggs, and six eggs from each batch were analysed for each variable. Results were expressed in absolute terms (as weight of component per egg) and as percentage of egg dry weight. Highly significant variations in all these aspects of egg composition were shown to occur between parent females. However, there was no significant correlation between the percentage of the original number of eggs which hatched and any one aspect of egg composition, except for a weak positive correlation (P<0.05) with the percentage of protein phosphorus in the eggs. Similarly, there was no significant correlation between the percentage of the original number of eggs which reached first feeding and any one aspect of egg composition except for weak positive correlations (P<0.05) with egg wet weight and with both the absolute level and the percentage of protein phosphorus in the egg. There was significant positive correlation (P<0.05) between the percentage of hatched eggs (alevins) surviving to first feeding and each of the following: egg wet weight, egg dry weight and absolute levels in the egg of bound lipid, precipitable protein and protein phosphorus. Egg batches with higher hatching percentage (>50%) differed significantly from those with zero hatching percentage in having (in absolute terms) higher egg weight, chorion weight, protein phosphorus (all P<0.001), egg dry weight, bound lipid and precipitable protein (all P<0.01) and (percentages) higher chorion weight (P<0.05) and protein phosphorus (P<0.001), and lower free and total lipid (both P<0.01) and iron (P<0.05). In a separate experiment to investigate the effects of allowing the eggs to be retained by the female within the abdominal cavity for increasing periods of time after ovulation, eggs were obtained from three females on three or four successive occasions 2–11 days apart. Although the above aspects of egg composition remained almost constant when the eggs were held in the female for up to 18 days after ovulation, the hatching percentage declined sharply within this period, in two females falling from over 90% to near zero. These results together indicate that the time of stripping of the eggs in relation to the date of ovulation is a much more significant determinant of egg quality than any of the chemical and physical aspects of egg composition which were investigated.     

Biochemical changes associated with overripening of the eggs of rainbow trout Salmo gairdneri Richardson

This study of the biochemical composition of mature unfertilised eggs of rainbow trout was undertaken to investigate normal intraspecific variation in egg composition between parent females, and to describe the biochemical changes which occur after ovulation during overripening in the parental abdominal cavity. The following aspects of egg composition were measured: wet and dry weight, chorion weight, free and bound lipid, protein phosphorus, lipid phosphorus, precipitable protein, calcium, and iron. All these were determined on single eggs, and six eggs were examined from each female. Ripe eggs, defined as those stripped within 3 days of ovulation, were obtained from 7 females. Overripe eggs, defined as those stripped 30 or more days after ovulation were obtained from 8 females. Results were expressed in absolute terms (mg or μg of component per egg) and relative terms (percentage of egg dry weight). In ripe eggs, there was significant variation between females in all these aspects of egg composition when expressed absolutely, and in all except lipid phosphorus when expressed relatively. In overripe eggs, there was significant variation in all these aspects between females when egg composition was expressed either absolutely or relatively. Comparison of ripe and overripe eggs showed that overripening was accompanied by the following significant changes expressed absolutely: increase in water content, free lipid, iron, calcium; decrease in bound lipid, precipitable protein, protein phosphorus, lipid phosphorus, total lipid and chorion weight; when expressed relatively there were increases in water content, free lipid, iron, calcium, total lipid and chorion weight, and decreases in bound lipid, precipitable protein, protein phosphorus and lipid phosphorus.     

Evaluation of Sperm-Activating Solutions in Atlantic Salmon Salmo salar Fertilization Tests

The use of saline solutions was tested to determine their efficacy as replacements for ovarian fluid as sperm activators and to eliminate variability encountered with the use of Ovarian fluid. We tested fertilization rate of semen from eight males on Atlantic salmon Salmo salar eggs after five sperm-activating solutions and a non-activating saline were substituted for ovarian fluid. We used solutions shown acceptable for use with other salmonid species. The six solutions tested were a non sperm-activating phosphate-buffered saline (PBS, 7.2 g/L NaCl, 1.48 g/L Na₂HPO₄, 0.43 g/L K H₂PO₄), a Tris buffer (6.99 g/L NaCl, 3.63 g/L Tris and 2.42 g/L glycine), a Borax buffer (12.2 g boric acid/L in solution 1, 76 g disodium tetraborate/L combined 100:118, then 1 L combined with 3.7 L water and 18 g NaCl), and three solutions of 9.25 g/L (125 mM) NaCl buffered to pH 6.0, 7.5, and 8.9. The latter five solutions activated sperm immediately on contact, while PBS required additional water to activate sperm. The PBS solution was the least effective (mean percent eyed eggs 37.6%) for egg fertilization. The mean percent eyed eggs for the other five saline solutions (range 78% to 86%) were not significantly different. Sperm from one male provided significantly lower egg fertilization (39.6%) when compared with the other seven males (67.2–87.4% egg fertilization). Percent egg fertilization was not related to number of live sperm cells per egg. Our results show that osmotically-balanced sperm-activation solutions, even those with a pH range from 6.0 to 8.9 provide adequate media for fertilization of Atlantic salmon eggs. Fertilization in a deactivation saline and water reactivation of sperm resulted in low egg fertilization.     

High fry production rates using post-thaw silver carp (Hypophthalmichthys molitrix) spermatozoa under farming conditions

Silver carp (Hypophthalmichthys molitrix) is not endemic to Cuba, and egg fertilization is totally artificial; males produce spermatozoa only between March and October. Cryopreservation of silver carp spermatozoa would reduce the number of males needed, minimize handling stress through less frequent stripping, and facilitate artificial propagation when eggs are available. The effects on motility and fry production from eggs fertilized with thawed sperm under farm-conditions were examined in this study. Five, seven and ten percent of dimethyl sulfoxide (DMSO), glycerol and methanol were tested as cryoprotectants. DMSO was a more suitable cryoprotectant than methanol or glycerol. The effect of equilibration time on the motility rate at 10% DMSO was evaluated. Hatching rates equal to the control (P>0.01) were obtained under farm conditions with frozen spermatozoa, stored even for a year in liquid nitrogen, with a final DMSO concentration of 10%. Cryopreservation offers a useful routine method for sperm storage and silver carp handling. To our knowledge, this is the first report on the production of fry from post-thaw silver carp spermatozoa under farming conditions.     

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica

Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g^-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish^-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g^-1 BW week^-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3^rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.     

Cryopreservation of Paddlefish Polyodon spathula Milt

Abstract.— A practical procedure for cryopreserving milt of paddlefish Polyodon sparhula was developed to obtain thawed spermatozoa that would fertilize eggs and permit hatching of normal larvae. Milt was mixed with a cryoprotectant medium containing DMSO (2.4 M) in a ratio of 3:1(milt: medium; final concentration of DMSO 0.6 M), stored in 5.0-mL freezing straws, and frozen in dry ice (15 min) and then in liquid nitrogen. A total of three replicates were made; the milt of a different male was used in each replicate. Motility of the thawed spermatozoa decreased to 50%-25% as compared to 100% motility of the fresh (control) spermatozoa. Hatching of paddlefish (16.3 ± 2.2%) from eggs fertilized with thawed spermatozoa was significantly lower ( P ≤ 0.01) than the hatch rate (90.8 ± 2.5%) for the control. It was suggested that an increase in viable motile spermatozoa to egg would result in better fertilization and hatching of paddlefish.     

锯缘青蟹卵膜变化与卵子附着研究

利用光镜和电镜观察了锯缘青蟹成熟卵及其受精后卵膜变化和卵子附着特征。锯缘青蟹成熟卵子卵膜有三层即外层、内层和质膜。卵子产出后,无论受精与否,外层和内层卵黄膜形成壳膜,与卵子附着有关。刚产出的卵子卵膜具有溶胶性和延展性,卵子与刚毛表面之间或卵子之间接触便粘在一起,由于刚毛摆动和卵子的重力作用,形成卵柄或卵索。卵子受精后,发生皮层反应形成受精膜。由壳膜和受精膜构成的孵化膜为初级卵膜,保护胚胎发育,直至     

Cryopreservation of sea bass (Dicentrarchus labrax) spermatozoa in experimental and production simulating conditions

A sperm cryopreservation protocol adapted from turbot, was tested on sea bass using either 250-μL straws or 1.5-mL cryovials. A dilution to 1/3 in Mounib s extender and a cooling rate of −65 °C·min⁻¹ allowed frozen sperm to recover an initial motility similar to that of fresh sperm at thawing; however, significant differences in motility (P < 0.001, n = 10 fish semen) were observed at further post-activation times, the motility decrease being faster in thawed sperm. At the experimental scale, triplicate inseminations of 2-mL aliquots (approximately 2 000 eggs) showed a significant fertility decay of thawed sperm compared to that of fresh sperm (P < 0.01, n = 12 fish semen) when a discriminating 35·10³ spermatozoa to egg ratio was applied. When 70·10³ and 200·10³ spermatozoa per egg were provided in the same experimental conditions, no significant difference appeared between the fertilisation rates of fresh and thawed sperm. In order to validate the procedure for production or cryobank purpose, a scaled-up protocol was established. Two and 50 mL batches of eggs (approximately 2·10³ and 50·10³ eggs, respectively) were inseminated in triplicate using either fresh or thawed individual sperms of 5 males with 200·10³ spermatozoa per egg. The mean fertility decreased by 23.5 % due to cryopreservation. This decline was explained by the loss of fertility of only one sperm, and only in large-volume conditions, probably due to the delay of use after thawing.     

Energy content and chemical composition of gilthead seabream, Sparus aurata L., eggs

Abstract. Gross energy, ash content and elemental composition of carbon, nitrogen and hydrogen in eggs of the gilthead seabream, Sparus aurata L., have been determined throughout several spawning seasons. Also, changes in the biochemical components during embryonic development have been analysed. Gross energy of seabream eggs was approximately 1 J per egg. Dry weight of individual eggs at fertilization varied from 39 to 50μg, the chorion accounting for about 20% of total dry weight. However, there were no differences in carbon, nitrogen, hydrogen and ash percentages. From fertilization to hatching eggs lost 7·2% of total energy (chorion not included). The possible relationship between egg energy content and recent emerged larvae is discussed.     

Saprolegnia diclina IIIA and S. parasitica employ different infection strategies when colonizing eggs of Atlantic salmon,Salmo salar L.

Here, we address the morphological changes of eyed eggs of Atlantic salmon, Salmo salar L. infected with Saprolegnia from a commercial hatchery and after experimental infection. Eyed eggs infected with Saprolegnia spp. from 10 Atlantic salmon females were obtained. Egg pathology was investigated by light and scanning electron microscopy. Eggs from six of ten females were infected with S. parasitica, and two females had infections with S. diclina clade IIIA; two Saprolegnia isolates remained unidentified. Light microscopy showed S. diclina infection resulted in the chorion in some areas being completely destroyed, whereas eggs infected with S. parasitica had an apparently intact chorion with hyphae growing within or beneath the chorion. The same contrasting pathology was found in experimentally infected eggs. Scanning electron microscopy revealed that S. parasitica grew on the egg surface and hyphae were found penetrating the chorion of the egg, and re‐emerging on the surface away from the infection site. The two Saprolegnia species employ different infection strategies when colonizing salmon eggs. Saprolegnia diclina infection results in chorion destruction, while S. parasitica penetrates intact chorion. We discuss the possibility these infection mechanisms representing a necrotrophic (S. diclina) vs. a facultative biotrophic strategy (S. parasitica).     

Some properties of bream Abramis brama L. sperm and its cryopreservation

Concentration and motility of spermatozoa, total protein content and its electrophoretic profile, glucose content, activity of aspartate aminotransferase (AspAT) and acid phosphatase (AcP) were assessed in 18 samples of semen from common bream Abramis brama L. males, which were hormonally stimulated to spermiation. Also, milt pooled from four donors was cryopreserved as pellets in vapours of liquid nitrogen (?80 °C) using four extenders (each with or without the addition of hen egg yolk). Mean spermatozoa concentration was 11.68 × 10⁹ mL^?1, and mean spermatozoa motility was about 60%. Protein content in seminal plasma was 2.08 mg mL^?1; both PAGE and SDS–PAGE showed considerable heterogeneity of protein fractions. Mean glucose content was over 11 mg%. AspAT and AcP activities were detected in both seminal plasma and spermatozoa extracts. As calculated to 1 × 10⁹ spermatozoa, AcP and AspAT activities were almost sixfold and 46-fold higher in spermatozoa than in seminal plasma respectively. In the best variant, cryopreservation attempts resulted in 66.6% of eyed embryos (compared with control fertilization) obtained after fertilization of eggs with cryopreserved semen.