Induction of diploid gynogenesis in turbot Scophthalmus maximus with left-eyed flounder Paralichthys olivaceus sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig's effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm(-2). At 15 degrees C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3 degrees C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1 degrees C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.

Development of expressed sequence tags from the bay scallop, Argopecten irradians irradians

The bay scallop, Argopecten irradians irradians, introduced from North America, has become one of the most important aquaculture species in China. Inan effort to identify scallop genes involved in host defense, a high-quality cDNA library was constructed from whole body tissues of the bay scallop. A total of 5828 successful sequencing reactions yielded 4995 expressed sequence tags (ESTs) longer than 100 bp. Cluster and assembly analyses of the ESTs identified 637 contigs (consisting of 2853 sequences) and 2142 singletons, totaling 2779 unique sequences. Basic Local Alignment Search Tool (BLAST) analysis showed that the majority (73%) of the unique sequences had no significant homology (E-value >= 0.005) to sequences in GenBank. Among the 748 sequences with significant GenBank matches, 160 (21.4%) were for genes related to metabolism, 131 (17.5%) for cell/organism defense, 124 (16.6%) for gene/protein expression, 83 (11.1%) for cell structure/motility, 70 (9.4%) for cell signaling/communication, 17 (2.3%) for cell division, and 163 (21.8%) matched to genes of unknown functions. The list of host-defense genes included many genes with known and important roles in innate defense such as lectins, defensins, proteases, protease inhibitors, heat shock proteins, antioxidants, and Toll-like receptors. The study provides a significant number of ESTs for gene discovery and candidate genes for studying host defense in scallops and other molluscs.

Effect of ultraviolet irradiation on sperm of the left-eyed flounder, Paralichthys olivaceus

In this study, at proper dosage of ultraviolet (UV) irradiation (180 sec: 36,000 erg/mm(2)), sperm chromosomes of left-eyed flounder, Paralichthys olivaceus, were inactivated, while spermatozoa maintained ability to move and inseminate eggs. Gynogenetic haploids were detected by morphological observation, chromosome counting, and flow cytometer analysis. The ultrastructure of treated sperm was observed under scanning electronic microscope (SEM) and transmission electronic microscope (TEM). The results showed that after being irradiated at lower dosage of irradiation (0-180 sec: 0-36,000 erg/mm(2)), the surface structure of spermatozoa was not affected by UV irradiation, while the inner structures including membrane system and karyoplasm denseness of treated spermatozoa were little changed. However, obvious changes were observed in their membrane system, mitochondria, and nucleus if the dosage of irradiation increased to 240 sec: 48,000 erg/mm(2) or 300 sec: 60,000 erg/mm(2). The sperm survival rates did not change at the lower dosages of the UV irradiation (0-180 sec: 0-36,000 erg/mm(2)) but decreased as the irradiation dosage increased. The motility of treated sperm was lower than that of control group in general but did not change with UV irradiation dosage increasing at the certain range of 0-300 sec: 0-60,000 erg/mm(2).

大西洋庸鲽Hippoglossus hippoglossus和大西洋鲑Salmo salar配子质量研究

种质问题是养殖健康发展的基础。在鱼类养殖中，卵子和精子的质量直接关系到受精、胚胎发育，仔稚鱼发育以及幼鱼生长等一系列过程。本论文针对大西洋庸鲽和大西洋鲑的配子质量进行研究。研究内容涉及大西洋庸鲽精子冷冻保存方法；促性腺激素释放激素类似物（GnRHa）使用对其精子冷冻保存效果、以及脂肪酸组成的影响；野生和驯养大西洋鲑卵子在脂肪酸、类胡萝卜素、矿物盐方面的差异比较。 精子冷冻保存通过提高对精子的利用效率，进而对于种质改良，推进鱼类养殖科研和生产具有重要意义。本实验建立了大西洋庸鲽精子大容量冷冻保存方法。八种抗冻剂冷冻保存实验结果表明：10% 及15% DMSO配以 HBSS 或KS 的抗冻剂组合冷冻保存效果最佳，4 mL体积冷冻保存可获得与1.6 mL同样的保存效果。 在繁殖季节后期注射GnRHa激素缓释剂，可获得质量稳定的大西洋庸鲽精液，将激素注射方法与精子冷冻保存方法相结合对于提高雄鱼利用率，扩大生产规模具有重要实用价值。本项研究分三个时间采集注射GnRHa激素后的雄鱼精子以及同期未注射激素的雄鱼精子，对所有精子样品使用同样的方法进行冷冻保存，检测冷冻保存后解冻精子的受精率与活力。结果表明，激素注射与否对于冷冻保存后精子的受精率和活力无显著影响，两类冷冻精液均达到鲜精水平。实验结果还表明，注射激素14天后的精子的密度显著的降低。说明GnRHa激素的使用可以显著降低精子密度，但不会影响精子的冷冻保存效果。 本相研究同时对注射GnRHa 缓释激素和未注射GnRHa 缓释激素的大西洋庸鲽精液脂肪酸成分进行分析，以检测该激素使用对精子生化组分的影响。结果表明激素的使用对在DHA (22:6n-3,二十二碳六烯酸)、EPA(20:5n-3,二十碳五烯酸)、AA(20:4n-6，花生四烯酸)等重要脂肪酸，不饱和脂肪酸、饱和脂肪酸以及n-3、n-6等重要种类的脂肪酸总量及其比例没有显著影响。精液脂肪酸中DHA含量最高，约占25%；PUFA约为44%。 作为世界性的重要养殖品种，野生和驯养大西洋鲑在形态、生化组成以及遗传 等方面表现出的差异被广泛关注。本论文，对野生和驯养大西洋鲑受精卵关键生化成分进行分析，通过与野生受精卵比较阐明驯养受精卵的质量状况，为亲鱼营养需求提供指导依据。本实验中野生配子和驯养配子的受精率没有显著差异，但重要脂肪酸组成、类胡萝卜素以及矿物盐含量都存在多方面显著差异。两类受精卵脂肪酸中含量最高的依次为18:1n-9（油酸）、DHA（二十二碳六烯酸）、16:0（棕榈酸）、EPA（二十碳五烯酸）。野生受精卵的单不饱和脂肪酸总量显著高于驯养受精卵，而多不饱和脂肪酸（PUFA）比例显著低于驯养的受精卵。在主要必需不饱和脂肪酸（EFA）中，DHA和EPA在野生受精卵中的比例高于驯养受精卵，AA（花生四烯酸）低于驯养受精卵。野生受精卵虾青素（Ax）的含量低于驯养受精卵而鸡油菌素（Cx）含量高于驯养受精卵。野生受精卵中多种矿物盐的含量（铝、铜、铁、硒和锌）含量显著高于驯养的受精卵。差别最大的为铜。诸多方面的差异表明，野生亲鱼与驯养亲鱼产出的卵子确实存在显著差异，因此关注亲鱼的营养极为重要。

A preliminary study on the mechanism of harmful algal bloom mitigation by use of sophorolipid treatment

in order to investigate a new method of mitigating the deleterious effect of harmful algal blooms (HABs), the inhibition of the glycolipid biosurfactant sophorolipid on three common harmful algae Alexandrium tamarense, Heterosigma akashiwo and Cochlodinium polykrikoides was studied. The optimum preparation condition for sophorolipid, the inhibition capability of sophorolipid and the interaction mechanism of sophorolipid on the three algal species were investigated. Results showed that sophorolipid prepared by extraction with ethyl acetate exhibited the most prominent inhibition effect and that storage time of one year had little influence on the inhibition effect of sophorolipid. The optimum concentration of 10-20 mg/l sophorolipid inhibited the motility of about 90% of the tested harmful algal cells without recovery. Investigation of the algicidal process revealed that sophorolipid induced ecdysis of A. tamarense, quick lysis of H. akashiwo and swelling of C. polykrikoides in a relatively short time. Investigation of the nucleotides showed that more than 15% of the nucleotides were released from the cytoplasm under the effect of 10-20 mg/l sophorolipid, indicating the irreversible damage on the cellular membrane, which resulted in the disintegration of the harmful algal cells. (C) 2004 Elsevier B.V. All rights reserved.