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ABSTRACT: The morphology of gametes and the fertilization biology of Japanese flounder Paralichthys olivaceus (Po), summer flounder Paralichthys dentatus (Pd) and their hybrids were examined. Multiple generations (two hybrids: Po♀× Pd♂ (F1) and Pd♀× Po♂; two backcrosses: F1♀× Po♂ and F1♀× Pd♂) were obtained by artificial insemination. Comparison of egg ultrastructure among Po, Pd and F1 showed the morphology of micropyle region and the distribution density of pores were species specific. There were c. 100-200 accessory openings around the micropyle in Po, but not in Pd and F1. The zona radiata thickness and number of parallel bands were similar between F1 and Po, which were different from Pd. Comparison of spermatozoa ultrastructure revealed a close relationship between Po and Pd. Cytologically, the six crosses obeyed normal fertilization and cleavage processes, and only one male pronucleus was observed in a fertilized egg, indicating a monospermic fertilization pattern. Analysis of the time distribution from fertilization to first cleavage revealed an obvious delay at pronucleus fusion in the Pd × Po cross. The delay might indicate some cytoplasmic-nuclear incompatibility during the process of fertilization.
Journal of Fish Biology 03/2012; 80(3):473-85. · 1.68 Impact Factor
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ABSTRACT: To describe the skeletal development and abnormalities in turbot Scophthalmus maximus, samples were collected every day from hatching to 60 days after hatching (DAH). A whole-mount cartilage and bone-staining technique was used. Vertebral ontogeny started with the formation of anterior haemal arches at 5·1 mm standard length (L(S) ) c. 11 DAH, and was completed by the full attainment of parapophyses at 16·9 mm L(S) c. 31 DAH. Vertebral centra started to develop at 6·3 mm L(S) c. 16 DAH and ossification in all centra was visible at 11·0 mm L(S) c. 25 DAH. The caudal fin appeared at 5·1 mm L(S) c. 11 DAH and ossification was visible at 20·6 mm L(S) c. 37 DAH. The onset of dorsal and anal fin elements appeared at 5·8 mm L(S) c. 15 DAH and 6·3 mm L(S) c. 16 DAH, respectively. Ossifications of both dorsal fin and anal fin were visible at 20·6 mm L(S) c. 37 DAH. The pectorals were the only fins present before first feeding, their ossifications were completed at 23·5 mm L(S) c. 48 DAH. Pelvic fins began forming at 7·2 mm L(S) c. 19 DAH and calcification of the whole structure was visible at 19·8 mm L(S) c. 36 DAH. In the present study, 24 types of skeletal abnormalities were observed. About 51% of individuals presented skeletal abnormalities, and the highest occurrence was found in the haemal region of the vertebral column. As for each developmental stage, the most common abnormalities were in the dorsal fin during early metamorphic period (stage 2), vertebral fusion during climax metamorphosis (stage 3) and caudal fin abnormality during both late-metamorphic period (stage 4) and post-metamorphic period (stage 5). Such research will be useful for early detection of skeletal malformations during different growth periods of reared S. maximus.
Journal of Fish Biology 03/2012; 80(3):486-502. · 1.68 Impact Factor
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ABSTRACT: Digestive enzyme activities were analysed in turbot (Scophthalmus maximus) from hatching until 60 days after hatching (DAH). Trypsin sharply increased to the climax at 17 DAH and decreased until 31 DAH followed by a stable level thereafter. Amylase was determined at 4 DAH, reached the maximum value at 19 DAH and declined sharply to 39 DAH and remained at a low level thereafter, suggesting the carbohydrate component should remain at a low level in formulated diets. Pepsin was detected at 9 DAH and increased to 34 DAH and then remained at a stable level. The above results revealed pancreatic enzymes are no longer main enzymes for food digestion after the formation of functional stomach. Leucine-alanine peptidase (Leu-ala) and alkaline phosphatase (AP) and leucine aminopeptidase N (LAP) were found in newly hatched larvae. Both AP and LAP activities markedly increased to 23 DAH, decreased abruptly to 50 DAH and increased gradually to 60 DAH. Leu-ala reached the plateau from 23 to 39 DAH, followed by a decline to 46 DAH and an increase until 60 DAH. The brush border membrane (BBM)-bound enzyme activities increased from 30% at 31 DAH to 81% at 38 DAH of the total activities, indicating the maturation of intestinal tract.
Fish Physiology and Biochemistry 09/2011; 38(3):715-24. · 1.53 Impact Factor
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ABSTRACT: The growth potential of turbot Scophthalmus maximus larvae and juveniles was studied using nucleic acid-based indices and protein variables. The experiment was carried out from 4 to 60 days post hatching (dph). A significant increase in instantaneous growth rate during metamorphosis and retarded growth rate during post-metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein all showed developmental stage-specific traits. The RNA:DNA ratio decreased up to 12 dph, then increased rapidly till 19 dph and fluctuated until 35 dph followed by a decline to the end. The RNA:DNA ratio was positively correlated with growth rate of juveniles during the post-metamorphic phase, whereas this ratio was not a sensitive indicator of growth during the pre-metamorphic phase and metamorphosis. The protein:DNA ratio showed a similar tendency to the RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of S. maximus performed mainly by hyperplasia from 4 to 12 dph and hypertrophy until 21 dph during the pre-metamorphic larval phase. Growth was dominantly hypertrophical from the early- to mid-metamorphosing phase and hyperplastic thereafter. The results show that the DNA content and protein:DNA ratio can evaluate growth rates of larval and juvenile S. maximus on a cellular level.
Journal of Fish Biology 08/2010; 77(3):512-25. · 1.68 Impact Factor
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ABSTRACT: The ice crystal formation is assumed as the most lethal factor for the failure of fish embryo cryopreservation and intracellular ice formation (IIF) plays a central role in cell injury during cooling. The objectives were to observe the morphological changes of red seabream (Pagrus major) embryo during the cooling-thawing process, and to examine the effect of cryoprotectant and cooling rate on the temperatures of oil globule ice formation (T(OIF)), extra-cellular ice formation (T(EIF)) and intracellular ice formation (T(IIF)) using cryomicroscope. After thawing, the morphological changes of embryos were observed and recorded by the video attachment and monitor under the microscope. During the cooling process, three representative phenomena were observed: oil globule gradually turned bright firstly, then the whole field of view flashed and the embryo blackened. Cooling rate affect the temperature of both extra- and intra-cellular ice formations. T(EIF) and T(IIF) at high cooling rate were much lower than that at low cooling rate. And the value of T(EIF)-T(IIF) increased from 0.45 to 11.11 degrees C with the increase of cooling rate from 3 to130 degrees C/min. Taken together, our results suggested that high cooling rate with proper cryoprotectant would be a good option for red seabream embryo cryopreservation.
Cryobiology 05/2009; 59(1):48-53. · 2.06 Impact Factor
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ABSTRACT: The objectives were to investigate the effect of cryoprotectants on the hatching rate of red seabream embryos. Heart-beat embryos were immersed in: five permeable cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), 1,2-propylene glycol (PG), and ethylene glycol (EG), in concentrations of 5-30% for 10, 30, or 60 min; and two non-permeable cryoprotectants: polyvinylpyrrolidone (PVP), and sucrose (in concentrations of 5-20% for 10 or 30 min). The embryos were then washed and incubated in filtered seawater until hatching occurred. The hatching rate of the embryos treated with permeable cryoprotectants decreased (P<0.05) with increased concentration and duration of exposure. In addition, PG was the least toxic permeable cryoprotectant, followed by DMSO and EG, whereas Gly and MeOH were the most toxic. At a concentration of 15% and 30 min exposure, the hatching rate of the embryos immersed in PG was 93.3+/-7.0% (mean+/-S.D.), however, in DMSO, EG, Gly, and MeOH, it was 82.7+/-10.4, 22.0+/-5.7, 0.0+/-0.0, and 0.0+/-0.0%, respectively. Hatching rate of embryos treated with PVP decreased (P<0.05) with the increase of concentration and exposure time, whereas for embryos treated with sucrose, there was no significant decrease in comparison with the control at the concentrations used.
Theriogenology 09/2008; 70(7):1086-92. · 1.96 Impact Factor
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ABSTRACT: The ice crystal formation is assumed as the most lethal factor for the failure of fish embryo cryopreservation and intracellular ice formation (IIF) plays a central role in cell injury during cooling. The objectives were to observe the morphological changes of red seabream (Pagrus major) embryo during the cooling–thawing process, and to examine the effect of cryoprotectant and cooling rate on the temperatures of oil globule ice formation (TOIF), extra-cellular ice formation (TEIF) and intracellular ice formation (TIIF) using cryomicroscope. After thawing, the morphological changes of embryos were observed and recorded by the video attachment and monitor under the microscope. During the cooling process, three representative phenomena were observed: oil globule gradually turned bright firstly, then the whole field of view flashed and the embryo blackened. Cooling rate affect the temperature of both extra- and intra-cellular ice formations. TEIF and TIIF at high cooling rate were much lower than that at low cooling rate. And the value of TEIF − TIIF increased from 0.45 to 11.11 °C with the increase of cooling rate from 3 to130 °C/min. Taken together, our results suggested that high cooling rate with proper cryoprotectant would be a good option for red seabream embryo cryopreservation.
Cryobiology.
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ABSTRACT: The objectives were to investigate the permeability of DMSO to red seabream (Pagrus major) embryos by capillary electrophoresis and the effects of DMSO concentrations (5 to 40 percent, volume basis) and immersion times (10, 30 and 60 min) on hatching rate and morphology. The results suggested the internal DMSO concentrations were positively related with the external concentrations and exposure times, while the hatching rate was negatively related. The hatching rate decreased drastically (less than 50 percent) after exposure in 35 percent, 20 percent and 15 percent DMSO for over 10, 30 and 60 min, respectively. In all groups, when hatching rate was greater 50 percent, the internal DMSO concentration was less than 2 percent, which was still insufficient for successful cryopreservation. Morphological changes indicated the chorion was permeable to the cryoprotectant. A sign of dehydration in yolk were observed, for a significant decrease in the maximal yolk sac diameter. However, further research was needed to investigate whether the DMSO permeated into the yolk.
Cryo letters 32(4):339-48. · 1.25 Impact Factor
