Identification of formalin-preserved eggs of red sea bream (Pagrus major) (Pisces: Sparidae) using monoclonal antibodies

Catches of important commercial fish such as red sea bream, flat fish, and yellowtail are decreasing in Japan. In order to sustain these species it is especially important that their distribution and biomass at all life stages are known. However, information on the early life stages of these species is limited because identifying the eggs and larvae of such fish is sometimes extremely difficult.

Influences of dietary lipid and phosphorus levels on retention and excretion of phosphorus and nitrogen in fingerlings red sea bream, Pagrus major

A laboratory based 2x3 factorial experiment was conducted for 12 weeks to investigate the influences of dietary lipid and phosphorus (P) levels on retention and excretion of phosphorus and nitrogen (N) in fingerling red sea bream. Two levels of lipid (210 and 260 g/kg) and three levels of phosphorus (17, 14 and 12 g/kgˉ¹) in the dry diets were tested. Duplicate groups of 25 red sea bream (average weight 3.74±0.07 g) per 60L glass tank were fed experimental diets three times a day near to satiation level at 22 to 28°C water temperature. A reduction in dietary fish meal from 500 to 300 g/kg dry diet, corresponding to a supplementation in both dietary lipid and P resulted in significant increase in both P and N retention which resulted in the reduction of their excretion by red sea bream. The overall results of the present study demonstrated that both lipid and phosphorus supplementation are necessary for developing less-polluting feed which in turn, reduce fish meal level in the diet of fingerling red sea bream. Further studies in this regard with different size and age groups of red sea bream are warranted.

Effect of cryoprotectants on hatching rate of red seabream (Pagrus major) embryos

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. (C) 2008 Elsevier Inc. All rights reserved.

Effect of storage time and cryoprotectant concentrations on the fertilization rate and hatching rate of cryopreserved sperm in red seabream (Pagrus major Temminck & Schlegel, 1843)

This study examined the effects of storage time and cryoprotectant concentrations on the post-thaw sperm of red seabream, Pagrus major. Sperm treated with 12%, 15%, 18% and 21% DMSO were cryopreserved for 10, 30, 60 and 360 days, and fertilization and hatching rates were analysed. For all groups, there were no differences in the fertilization rates and hatching rates between sperm cryopreserved for < 60 days and fresh sperm (98.8 +/- 0.8%, 96.4 +/- 1.3%). However, for sperm cryopreserved for 360 days, both fertilization rates (88.6 +/- 3.0% to 7.0 +/- 1.9%) and hatching rates (79.4 +/- 7.2% to 3.3 +/- 0.8%) decreased drastically. Furthermore, the cryoprotectant concentrations affected sperm quality significantly (P < 0.05). When cryopreserved for 360 days, sperm treated with 15% DMSO obtained the best results compared with other concentrations. We suggest that 15% DMSO may be an effective cryoprotectant for long-term sperm cryopreservation of red seabream.

Cadmium toxicity to embryonic-larval development and survival in red sea bream Pagrus major

At 18 degrees C and 33 psu, 24 and 48 h LC50 values of cadmium (Cd) for red sea bream Pagrus major embryos were 9.8 and 6.6 mg l(-1), respectively, while 24,48, 72, and 96 h LC50 values for larvae were 18.9,16.2, 8.0, and 5.6 mg l(-1), respectively, indicating that embryos were more sensitive to Cd toxicity than larvae. Cd concentrations at >= 0.8 mg l(-1) led to low hatchability (0-90% in >= 0.8 mg l(-1) solutions vs. 97-100% in lower ones), delay in time to hatch, high mortality (38-100% vs. 1-10%), morphological abnormality (42-100% vs. 1-10%), reduced length (3.55-3.60 vs. 3.71-3.72 mm) in the embryos and larvae. They were Cd concentration dependent and potential biological significant endpoints for assessing the risk of Cd to aquatic organisms. Heart beat and yolk absorption of the larvae were significantly inhibited at some high concentrations but they were not as sensitive as other endpoints to Cd exposure. (C) 2008 Elsevier Inc. All rights reserved.

Extra- and intra-cellular ice formation of red seabream (Pagrus major) embryos at different cooling rates

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 to 130 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. (C) 2009 Elsevier Inc. All rights reserved.

Molecular cloning and expression of interleukin 1beta (IL-1 beta) from red seabream (Pagrus major)

The interleukin 1beta (IL-1beta) cDNA was cloned from the red seabream (Pagrus major) by homology cloning strategy. A cDNA fragment was amplified by PCR using two degenerated primers, which were designed according to the conserved regions of other known IL-1beta sequences, and elongated by 3' ends and 5' ends RACE PCR to get the full length coding sequence of red seabream IL-1beta (RS IL-1beta). The sequence contained 1252 nucleotides that included a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 410 bp and an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28.6 kD and putative isoelectric point pI of 5.29. The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature, but lacked the signal peptide and the clear ICE cut site, which were common in other nonmammalian IL-1beta genes. The RS IL-1beta had the highest homology with piscine IL-1beta according to phylogenetic tree analysis. The transcript expression was detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen in the pathogen challenged and healthy red seabream by RTPCR. Results showed that the RS IL-1beta mRNA was constitutively expressed in most of the tissues both in stimulated and un-stimulated fish, and the expression could be enhanced by pathogen challenging.

Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFa transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, Ever, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

真鲷（Pagrus major）胚胎超低温损伤机理研究

鱼类胚胎由于其自身结构特征：体积大、含水量高、多室结构等，迄今超低温保存尚未成功。超低温保存过程中所造成的冷冻损伤是制约鱼类胚胎超低温保存成功与否的关键，具体表现为渗透压影响、抗冻剂毒性、冰晶损伤等。系统研究并阐明鱼类胚胎冷冻损伤机理，是成功建立鱼类胚胎超低温保存技术的基础。本论文主要针对胚胎对渗透压的耐受性、抗冻剂对胚胎的渗透性、降温速率对胚胎内外冰晶形成温度的影响等冷冻损伤机理进行了系统研究，主要研究结果如下： 1.通过检测胚胎在不同浓度人工海水（0%、25%、50%、75%、1×、2×、3×、4×，渗透压范围0~3740 mOsm/kg）中的孵化率，确定了真鲷不同发育时期胚胎对渗透压的耐受范围，以及心跳期胚胎浸泡不同时间对渗透压的耐受范围。结果显示：①真鲷2-4细胞期、原肠期、10-14体节期胚胎、心跳期和出膜前期胚胎孵化率>50%时渗透压的范围依次为：919~1391 mOsm/kg、919~1391 mOsm/kg、462 ~1391 mOsm/kg、232~1878 mOsm/kg和692~1391 mOsm/kg，表明心跳期胚胎对渗透压变化的耐受范围最广；②在不同浓度人工海水中分别浸泡10 min、30 min、1 h、5 h和10 h后，真鲷胚胎孵化率无显著变化的渗透压范围分别为0~2804 mOsm/kg、0~1878 mOsm/kg、232~1391 mOsm/kg、232~1391 mOsm/kg和919~1391 mOsm/kg；结果表明心跳期胚胎对渗透压的耐受范围随浸泡时间的延长而减小。 2.采用毛细管电泳技术检测胚胎内部DMSO的浓度，并且分析了胚胎孵化率和胚胎内部DMSO的浓度随浸泡时间变化与外部抗冻剂的关系。结果表明胚胎孵化率随胚胎外部抗冻剂溶液浓度和浸泡时间的增加而降低；胚胎内部DMSO浓度随胚胎外部抗冻剂溶液浓度和浸泡时间的增加而增加。对胚胎孵化率（y1）随抗冻剂溶液浓度（x）的变化进行一元三次多项式回归，当浸泡时间分别为10 min、30 min和60 min时，回归方程依次为：y1 = -2832.7x3 + 575.01x2 - 37.011x + 99.641（R2 = 0.9722）；y1 = 30288x3 - 16322x2 + 2077.3x + 27.603（R2 = 0.9876）；y1 = 16052x3 - 5985.2x2 - 32.696x + 119.6（R2 = 0.9124）。对胚胎内部DMSO浓度（y2）随抗冻剂溶液浓度（x）的变化进行回归，当浸泡时间分别为10 min、30 min和60 min时，回归方程依次为：y2 = 0.2584e6.7294x（R2 = 0.9876）；y2 = 0.2521e10.964x（R2 = 0.9644）；y2 = 0.4054e10.95x（R2 = 0.8954）。 3. 利用低温显微镜观察了不同降温速率（20、40、60、80、100、120℃/min）对胚胎内外冰晶形成温度的影响。胚胎外部冰晶形成温度（TEIF）随降温速率的增加显著下降，在降温速率大于80℃/min之后，TEIF随降温速率增加而降低的幅度减小；胚胎内部冰晶形成温度（TIIF）在降温速率小于80℃/min 时随降温速率的升高而降低，在降温速率大于80℃/min 时随降温速率的升高而升高；胚胎内外冰晶形成温度差值（TEIF - TIIF）在降温速率小于80℃/min时随降温速率的升高而增大，在降温速率大于80℃/min时随降温速率的升高而减小。 4. 在低温显微镜下观察了真鲷胚胎低温保存中有复活胚胎记录的保存方法在冷冻解冻过程中的冰晶形成过程，结果表明：①在冷冻过程中，玻璃化法冷冻的胚胎的内部冰晶形成温度（-53.70，-64.33℃）显著低于程序降温法（-17.51，-21.40℃）；而且在玻璃化法冷冻的胚胎内部冰晶形成温度高于外部冰晶后形成（-70.30℃），程序降温法中则相反，胚胎内部冰晶形成温度显著低于外部冰晶形成温度（-4.93，-5.00℃）；玻璃化法中，40%PG冷冻的胚胎外部溶液出现玻璃化现象，其他组均未出现；②在解冻过程中，各组均出现重结晶现象；解冻后，玻璃化法的胚胎完整率（62.82%）远高于程序降温法（9.21%）。

Flow cytometry and ultrastructure of cryopreserved red seabream (Pagrus major) sperm

The objectives were to assess motility, fertilizing capacity, structural integrity, and mitochondrial function in fresh versus frozen-thawed (15% DMSO was used as a cryoprotectant) sperm from red seabrearn (Pagrus major). Mean (+/- S.D.) rates of motility, fertilization and hatching of frozen-thawed sperm were 81.0 +/- 5.4, 92.8 +/- 1.9, and 91.8 +/- 5.2%, respectively; for fresh sperm, they were 87.5 +/- 7.7, 95.8 +/- 2.4, and 93.8 +/- 4.2%. Although motility was lower in frozen-thawed versus fresh sperm (P < 0.05), there was no effect (P > 0.05) of cryopreservation on fertilization or hatching. Based on scanning and transmission electron microscopy, 77.8 +/- 5.6% of fresh sperm had normal morphology, whereas for frozen-thawed sperm, 63.0 +/- 7.2% had normal morphology, 20.6 +/- 3.1% were slightly damaged (e.g. swelling or rupture of head, mid-piece and tail region as well as mitochondria), and 16.4 +/- 4.2% were severely damaged. Sperm were stained with propidium iodide and Rhodamine 123 to assess plasma membrane integrity and mitochondrial function, respectively, and examined with flow cytometry. For fresh sperm, 83.9% had an intact membrane and functional mitochondria, whereas for frozen-thawed sperm, 74.8% had an intact membrane and functional mitochondria, 12.7% had a damaged membrane, 9.9% had nonfunctional mitochondria, and 2.6% had both a damaged membrane and nonfunctional mitochondria. In conclusion, ultrastructure and flow cytometry were valuable for assessment of frozen-thawed sperm quality; cryopreservation damaged the sperm but fertilizing ability was not significantly decreased. (c) 2007 Elsevier Inc. All rights reserved.

Use of computer-assisted sperm analysis (CASA) to evaluate the quality of cryopreserved sperm in red seabream (Pagrus major)

In the present study, the quality of post-thaw sperm of red seabream Pagrus major frozen with 6-24% DMSO was investigated. The motility, average path velocity and fertilizing capacity of fresh and their corresponding post-thaw sperm were examined for evaluation of the post-thaw sperm motion characteristics and its association with fertilizing capacity. An analysis of sperm motility before and after cryopreservation has been performed using computer-assisted sperm analysis (CASA). For post-thaw sperm frozen with 12-21% DMSO, the percentages of motile sperm were not significantly (P > 0.05) changed 10 s after activation. Moreover, the main motility pattern and swimming velocity of the motile post-thaw sperm were not significantly (P > 0.05) changed and the progressive linear motion was still the dominant pattern. However, the total motility of post-thaw sperm (72.3 +/- 6.3%) 30 s after activation was (P < 0.05) lower than the corresponding fresh sperm (82.7 +/- 7.2%). Additionally, the fertilizing capacity of post-thaw sperm was investigated with a standardized sperm to egg ratio 500:1. There is a linear regression relationship between the percentage of motile post-thaw sperm and fertilizing capability. These data demonstrate that 12-21% DMSO can provide good protection to the sperm during the freezing-thawing process. (c) 2006 Elsevier B.V. All rights reserved.

Effect of cryoprotectants on hatching rate of red seabream (Pagrus major) embryos

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. (C) 2008 Elsevier Inc. All rights reserved.

Effect of long-term cryopreservation on physiological characteristics, antioxidant activities and lipid peroxidation of red seabream (Pagrus major) sperm

The aim of this study was to determine the effect of long-term cryopreservation on physiological characteristics, the antioxidant activities and lipid peroxidation of red seabream sperm which were respectively cryopreserved with 15% dimethylsulfoxide (Me2SO) for 1 month, 13 months, 26 months, 48 months and 73 months. The motility and fertility of post-thaw sperm decreased with the storage time going on. The highest motility (87.67 +/- 2.52%) was obtained in sperm cryopreserved for 1 month and the lowest (50.67 +/- 5.31%) was in sperm for 73 months. There were no significant differences (p < 0.05) in fertilization rates of sperm cryopreserved for 1 month (71.33 +/- 8.84%), 13 months (69.22 +/- 1.02%) and 26 months (60.33 +/- 2.33%); however, the sperm fertility decreased significantly for 48 months (47.22 +/- 3.89%) and 73 months (39.56 +/- 0.69%) storage. In addition, superoxide dismutase (SOD) activities of sperm were at a stable level for less than 26 months storage, then, decreased significantly after 48 months storage. Catalase (CAT) activities of sperm cryopreserved for 13 months, 26 months, 48 months and 73 months were significantly lower than that for 1 month. There were no significant differences in the malondialdehyde (MDA) level of sperm for less than 13 months storage. After 26 months storage, the concentration of MDA increased significantly, and the highest concentration (3.22 +/- 0.05 nmol/mgprot) was obtained in 73 months storage sperm. (C) 2010 Elsevier Inc. All rights reserved.

An efficient methodology for cryopreservation of spermatozoa of red seabream, Pagrus major, with 2-mL cryovials

The aim of this study was to optimize the cryopreservation protocols for the sperm of red seabream, Pagrus major. The 2-mL cryovials and programmable freezer were employed for cryopreservation. Six extenders, six cryoprotectants in various concentrations ranging from 6 to 20% (v/v), four cooling rates, and three thawing temperatures were evaluated by postthaw sperm motility and fertility. The ratio of sperm to egg for postthaw sperm fertilization trials was experimentally standardized and was optimal at 500:1. The best motility of postthaw sperm (79.4 +/- 4.7% to 88.6 +/- 8.0%), fertilization rates (89.6 +/- 2.9 to 95.6 +/- 1.9%), and hatching rates (85.3 +/- 5.1% to 91.4 +/- 4.3%) were achieved when Cortland extender, dimethyl sulfoxide (15, 18, and 20%) or ethylene glycol (9, 12%) as cryoprotectants, 20 C/min as the cooling rate, and 40 C as the thawing temperature were employed. Moreover, the results on embryonic development were not significantly different between cryopreserved sperm and fresh sperm during incubation process. In conclusion, these methods of cryopreservation of red seabream sperm are suitable for routine aquaculture application and preservation of genetic resources.

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO + 10% PG + 10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6 +/- 16.7% (mean +/- S.D.) and 77.8 +/- 15.5%, were achieved by the straw vitrifying method (20.5% DMSO + 15.5% acetamide + 10% PG, thawing at 43 degrees C and washing in 0.5 M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5 M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation. (C) 2007 Elsevier Inc. All rights reserved.