The presence of uric acid, an antioxidantive substance, in fish seminal plasma

High concentrations of uric acid in seminal plasma of a range of teleost fish species are reported for the first time. Concentrations of urate amounted to 223.4; 121.9–130.0, 355.9, 735.6, 124.0, 192.7 and 148.0 M for rainbow trout (Oncorhynchus mykiss), yellow perch (Perca flavescens), muskellunge (Esox masquinongy), Northern pike (Esox lucius), carp koi (Cyprinus carpio), bream (Abramis brama), and tench (Tinca Tinca), respectively. Uric acid is an important anti-oxidant, therefore, it has a potential role in protection of fish spermatozoa against oxidative damage.     

Characterization of protease inhibitors of seminal plasma of cyprinids

Anti-proteinase activity was demonstrated in the seminal plasma of cyprinid fish species (bream, chub, ide, dace, asp, goldfish, roach, common carp) using electrophoretic techniques combined with a detection method based on inhibition of bovine trypsin. We found species-specific protease inhibitors in the seminal plasma of cyprinids. At least three bands of protease inhibitors with different migration rates could be identified by native PAGE. Higher variability was characterized for bands with slower migration rates. Visualization of inhibitors after SDS-PAGE under non-reducing conditions allowed estimation of their molecular weights. Apparent molecular weights were within the range of 51–59 and 47–54 kDa for the bands with slower and moderate migration rates, respectively. The molecular weight of fast migration bands for roach and common carp were estimated to 23 and 30 kDa, respectively. Inhibitors of common carp seminal plasma differed in their affinity toward serine proteases. Three inhibitors in common carp seminal plasma could be visualized using cod and bovine trypsin, but only two inhibitors (of high molecular weight) were recognized with chymotrypsin. There were differences in anti-proteinase activity and seminal plasma protein concentration in relation to the origin of common carp seminal plasma (breeding lines) and time of milt collection (spawning vs. post-spawning season).     

Antifreeze proteins in the urine of marine fish

Several species of marine teleosts have evolved blood plasma antifreeze polypeptides which enable them to survive in ice-laden seawater. Four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence and secondary structure are currently known. Although all of these antifreezes are relatively small (2.6–33 kd) it was generally thought that they were excluded from the urine by a variety of glomerular mechanisms. In the present study antifreeze polypeptides were found in the bladder urine of winter flounder (Pseudopleuronectes americanus), sea raven (Hemitripterus americanus), ocean pout (Macrozoarces americanus) and Atlantic cod (Gadus morhua). Since the plasma of each of these fish contains a different antifreeze class it would appear that all four classes of antifreeze can enter the urine. The major antifreeze components in the urine of winter flounder were found to be identical to the major plasma components in terms of high performance liquid chromatography retention times and amino acid composition. It is concluded that plasma antifreeze peptides need not be chemically modified before they can enter the urine.     

人工养殖施氏鲟和小体鲟精浆微量元素分析

采用原子吸收分光光度法测定了施氏鲟(Acipenserschrenckii)和小体鲟(Acipenserruthenus)的精浆中7种微量元素(K、Na、Mg、Zn、Cu、Fe、Mn)的含量。施氏鲟分别取自宜昌和荆州,小体鲟取自宜昌。分析结果显示,不同种类和产地的鲟精浆中微量元素含量有差异,其中2条不同产地施氏鲟精浆Zn含量有显著性差异(P<0.05);施氏鲟和小体鲟精浆Na、K含量与我国主要淡水养殖鱼类有很大差异,在2种鲟精浆中Na与K浓度之比为10～14。     

Hemolysis interference in measuring fish plasma biochemical indicators

The present study aimed to determine hemolysis interference in measurement of plasma biochemical parameters in fish specimens. For this purpose, blood samples were harvested from 24 Huso huso juveniles. After centrifugation, each plasma sample was divided into seven portions to make seven levels of hemolysis. Hemolysis was induced by addition of different percentages of the corresponding whole blood [0 (non-hemolyzed control group), 1, 3, 5, 10, 15, and 20% of whole blood]. Albumin, total protein, calcium, phosphorus, glucose, sodium, potassium, chloride, alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT) were measured in different samples. Results showed that plasma albumin, ALP, potassium, and AST significantly increased (more than 200% at the highest hemolysis level) in the hemolyzed samples. Also, plasma total protein and phosphorus showed significant elevation in the hemolyzed samples (more than 70% at the highest hemolysis level). Plasma glucose, calcium, chloride, and ALT showed narrow but significant increase in hemolyzed samples (11.8–35.2% at the highest hemolysis level). Plasma sodium showed no significant changes in the hemolyzed samples. In conclusion, the present results show that hemolysis markedly affects plasma parameters levels, which interferes with plasma results interpretation. Therefore, analysis of hemolyzed samples should be avoided or the results should be interpreted with caution. This study encourages further investigations to develop methods for omitting the effects of hemolysis by sample blanking and/or presenting correction coefficients for measurement of plasma parameters in samples with different levels of hemolysis.     

圆口铜鱼精浆的理化性质分析

对圆口铜鱼(Coreius guichenoti)精浆离子和氨基酸成分及精液生理特性进行了检测分析。结果显示,圆口铜鱼精液pH值为7.3,呈弱碱性;精液浓度为39.7%,精子密度为5.3×10~9个/mL;精浆离子以Na~+含量88.7 mmol/L最高,其次是K~+,之后为Ca~(2+)、Mg~(2+)、Zn~(2+)、Cu~(2+);精浆水解氨基酸总量为2 872.69μmol/100mL,其中以脯氨酸含量最高,蛋氨酸、酪氨酸和组氨酸含量最低。该结果填补了圆口铜鱼繁殖生物学的相关数据,为圆口铜鱼规模化人工繁育提供了基础资料。     

Identification and elimination of mycoplasmas in fish cell line cultures

Mycoplasma contamination was identified in 11 cell lines derived from both coldwater and warmwater fish using DNA staining, microbiological culture and ELISA test detection methods. Acholeplasma laidlawii and Mycoplasma arginini were present as single or combined infections. Treatment of cells over three to six serial subcultivations with the addition of 0.5 μg m^?1 mycoplasma removal agent (MRA) or 10 μg m^?1 ciprofloxacin to culture growth medium effectively eliminated all contaminants except for one instance of M. arginini infection.     

鱼类利用脂肪节约蛋白的研究进展

通常,鱼类由饲料中摄入的部分蛋白是作为能量来源而被消耗掉的。如果饲料中脂肪含量适当增加,则可以减少蛋白的比例,从而降低饲料成本,这一研究已经成为国际水产养殖领域的热点。国外学者对不同的鱼类进行了大量的尝试性研究,结果表明多种鱼具有利用脂肪节约蛋白的能力。脂肪节约蛋白对鱼类的生长摄食状态、机体的营养组成、免疫功能等具有明显的影响。介绍了脂肪和蛋白之间的转化及能量关系,综述了脂肪节约蛋白对鱼体的各种影响,并对该研究方向存在的问题和发展前景提出了一些建议。     

基于鱼体背部弯曲潜能算法的四种主养鱼类识别

以四种主养淡水鱼鲫(Carassius auratus)、草鱼(Ctenopharyngodon idellus)、团头鲂(Megalobrama amblycephala)、鲤(Cyprinus carpio)为例,提出一种基于图像处理技术的鱼种类自动识别方法。首先通过鱼体信息采集系统获取待识别鱼体图像,并对其进行灰度化与二值化,得到鱼体轮廓信息;然后采用邻域边界算法对鱼体的轮廓进行提取,根据轮廓曲线建立鱼体背部轮廓数学模型;最后根据轮廓模型,采用鱼体背部弯曲潜能算法对不同种类鱼体样本的背部弯曲潜能值进行计算和聚类统计,得到不同鱼类样本的背部弯曲潜能值分布区间,从而通过比对待识别鱼体背部弯曲潜能值的区间实现对不同鱼类的自动识别。对四种主养鱼类的实验测试结果表明,对团头鲂的识别精度可以达到100%,对鲫、鲤和草鱼的识别精度达到96%,基本上能准确实现四种鱼体的分类识别,具有较好的实际应用价值。     

Immunochemical relationships of cytochrome P4503A-like proteins in teleost fish

Multiple P450 proteins have been purified from several teleost species, including rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chrysops) and Atlantic cod (Gadus morhua). Identity, relationships and/or functions have been established in these fish species for the cytochrome P4501 As. Information about the structure, function, regulation and relationships of other piscine cytochrome P450 (CYP) proteins is sparse. In the present study we have focused on constitutively expressed CYP forms, P450con and LMC5 isolated from rainbow trout, P450A from scup, and P450b from Atlantic cod, and we consider evidence for the relationship of these proteins to mammalian members of the CYP3A subfamily. Reciprocal western blot analysis shows that P450con and LMC5, isolated from rainbow trout in two different laboratories, are closely related and ostensibly identical proteins. These trout proteins show specific reciprocal cross-reactivity with scup P450A, and polyclonal antibodies (PAb) to the trout and scup proteins both recognize cod P450b, indicating that rainbow trout P450con/LMC5, scup P450A and cod P450b are immunochemically-related proteins. In analyses of liver microsomes of trout, scup and cod, PAb to trout P450con/LMC5 and scup P450A recognize only bands that are identical in migration to the CYP proteins purified from these species, and which were used as immunogens. These CYP proteins purified from fish are each immunochemically-related to mammalian CYP3A proteins, showing recognition by PAb to human CYP3A4 and to rat CYP3A1. PAb to the mammalian CYP3As also recognize the same bands in liver microsomes from these fish species as seen by PAb to the fish proteins. These results strongly suggest that these fish proteins are members of theCYP3 gene family and probably theCYP3A subfamily. Although sequence analysis is required before their designation in the CYP3A subfamily can be confirmed and specified, we refer to these as CYP3A-like. Immunoblot analyses of hepatic microsomes from other fish species with PAb to scup P450A and trout P450con show that multiple CYP3A-like proteins are expressed in liver of several species, including killifish (Fundulus heteroclitus) and winter flounder (Pleuronectes americanus). Important questions still remain to be addressed concerning CYP3A structure, multiplicity, physiological function, regulation and metabolism of endogenous as well as exogenous substrates in fish.Part of this study was presented at the 10th International Symposium on Microsomes and Drug Oxidations. Toronto, Canada, July 18–21, 1994.     

Low seminal plasma buffering capacity corresponds to high pH sensitivity of sperm motility in salmonids

The buffering capacity of air-equilibrated seminal plasma from the chinook salmon Oncorhynchus tshawytscha and steelhead Oncorhynchus mykiss were determined. Buffering capacities were significantly less at pH values at and above 7.3 than over the range 6.0 to 7.0 and were particularly low between pH values of approximately 7.5 to 8.5. The potential of chinook salmon sperm to become motile upon addition to water was highly sensitive to pH. Only very low levels of motility were demonstrable when sperm had been maintained at pH values at or below 7.5 prior to water exposure while maximum motility was noted when sperm had been maintained at pH values of about 8.0 and above. Thus, the potential for motility appeared especially sensitive to that pH range over which seminal plasma exhibited its lowest buffering capacity. Buffering capacity, protein content and inorganic phosphate content of chinook salmon and steelhead seminal plasmas were less than those of corresponding blood plasma or ovarian fluid suggesting that seminal plasma is unique among salmonid body fluids in its composition and low buffering capacity. This low buffering capacity is likely to facilitate the role of the sperm duct epithelium in semen pH regulation and thus, in the sperm's acquisition of the potential for motility upon exposure to water. This revised version was published online in August 2006 with corrections to the Cover Date.     

超低温冷冻对俄罗斯鲟精浆和精子酶活性的影响

研究了超低温冷冻保存(-196℃)对俄罗斯鲟(Acipenser gueldenstaedti)精浆和精子中总三磷酸腺苷酶(AT-Pase)、琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)、肌酸激酶(CK)等酶活性的影响,并分别测定了冷冻前后俄罗斯鲟精浆和精子中酶的活性。结果显示,经过超低温冷冻保存后,俄罗斯鲟精子活力下降,精子内各酶活性均显著降低,添加冷冻保护液组精子中总ATPase、SDH、LDH和CK的活性分别从(198.47±14.43)U/mL、(30.00±2.65)U/mL、(6 982.29±24.32)U/L和(1.94±0.05)U/mL下降至(110.19±2.32)U/mL、(16.33±2.08)U/mL、(5 122.93±195.07)U/L和(1.49±0.14)U/mL。未添加抗冻剂组则分别下降至(2.25±0.33)U/mL、(11.67±0.58)U/mL、(4 488.04±78.33)U/L和(1.16±0.02)U/mL;精浆中酶的活性均显著升高,添加冷冻保护液组精浆总ATPase、SDH、LDH和CK活性分别从(12.70±0.57)U/mL、(7.50±0.71)U/mL、(2017.26±116.81)U/L和(2.93±0.59)U/mL升高至(92.49±5.18)U/mL、(13.33±0.58)U/mL、(3 688.97±172.67)U/L和(4.39±0.24)U/mL,未添加抗冻剂组则分别上升至(200.27±12.97)U/mL、(24.67±3.06)U/mL、(6 124.40±329.14)U/L和(5.20±0.16)U/mL。结果表明,超低温冷冻对俄罗斯鲟精浆和精子中酶活性及精子活力均有较大影响。     

南极鱼抗冻蛋白功能和进化及其应用研究进展

南极海域在大约2000万年降到了零度以下,在大多数鱼类由于无法适应极端寒冷环境而灭绝的情况下,长期生活在极端寒冷、氧气充足的南极海域中的南极鱼亚目(Notothenioids)鱼类由于发生适应性进化而存活下来,并且达120多种类。南极鱼在生化和生理等方面发生了众多适应性改变,抗冻蛋白的产生是其中最重要的适应性特征之一。目前在极地鱼类中发现抗冻蛋白有5种类型,包括抗冻糖蛋白(AFGP)和4种抗冻蛋白(AFP I、AFP II、AFPIII和AFPIV)。虽然这些抗冻蛋白的起源和进化有所不同,但他们都具有抗冻的功能。作为一类能够抑制冰晶生长的蛋白质,南极鱼抗冻蛋白能使鱼的体液冰点降低至–2.2℃,所以其体液在海水冰点之下仍能保持流动性,避免冷冻损伤的发生。由于抗冻蛋白的热滞活性、冰重结晶抑制等天然活性,使其在食品、农业、医药等领域具有良好的应用前景。本文将对南极鱼抗冻蛋白的起源、进化、功能、应用和目前存在的问题进行概括性的综述。     

Total and free thyroid hormones in plasma of tropical marine teleost fish

Plasma levels of L-thyroxine (T₄) and 3,5,3-triiodo-L-thyronine (T₃) and the percentage of plasma T₄ and T₃ present in the free (dialyzable) form (%FT₄ and %FT₃) were measured in 16 species (11 families) of tropical marine teleosts from an inshore Barbados reef. Mean plasma T₄ varied from 0.2 ng/ml to 42 ng/ml; mean plasma T₃ varied from < 0.2 ng/ml to 50 ng/ml. The highest T₄ and T₃ levels were recorded in parrot-fish and the lowest levels in filefish. The %oFT₄ and %FT₃ varied from 0.05–3.41%. Estimated levels of plasma free T₄ and free T₃ levels ranged from 0.4–466 pg/ml. The extremely wide inter- and intra-species ranges in levels of free T₄ and T₃ do not support a previous suggestion, based on temperate freshwater salmonid species, that free T₄ and T₃ levels in fish may fall within a relatively range narrow comparable to that of homeothermic vertebrates.     

美国金鳟和日本鳗鲡精浆的化学组成和渗透压

本研究采用原子吸收分光光度法(AAS)和气相渗透压仪测定了人工养殖美国金鳟和日本鳗鲡精浆的化学组成和渗透压。研究结果表明,Na+、K+是精浆中的主要离子,可能在维持精浆渗透压和精子活动能力中有重要的作用。人工养殖美国金鳟与日本鳗鲡相比,精浆离子含量各项指标除K含量较高之外(P<0.01),其它各项指标均无显著性差异。本实验的两种鱼与其它种类海淡水鱼类比较,各种离子含量均较高;美国金鳟精浆渗透压值与其它鱼类相似,均保持在300 mOsm/kg的水平。     

Seasonal changes in the biochemistry of seminal plasma and sperm motility in the ocean pout,Macrozoarces americanus

Sperm motility, pH and osmolality of seminal plasma varied throughout the reproductive season spanning the period from June to September. Initially, sperm motility was low, peaked in July and August and then fell again towards the end of the spawning season. While the pH of seminal plasma increased from pH 7.4 to 7.9 during the period of spermiation, the average seasonal pH (7.78 ± 0.03) remained close to an experimentally determined optimum pH range for ocean pout sperm motility (pH 8–9). Likewise, although the values for seminal plasma osmolality fell during the reproductive season, from 416–339 mmol kg^-1, the average osmolality value 356 ± 3 was within the optimum for sperm motility (300–400 mmol kg^-1). In comparing fluctuations in sperm motility with the biochemical composition of ocean pout seminal plasma during the spawning season, this analysis showed that increased Mg⁺⁺ levels were correlated with the summer period of maximum sperm motility. A seasonal decline in Na⁺ and Cl⁻ ion levels was reflected in lower seminal plasma osmolality values.     

Sperm motility and seminal plasma characteristics in Barbus sharpeyi (Günther, 1874)

Spermatozoa concentration, ionic composition, osmolality, glucose and total protein contents of seminal plasma and sperm motility were determined in Barbus sharpeyi (Cyprinidae, Teleosotei). Spermatozoa concentration ranged from 9.77 to 20.20 × 10⁹ spermatozoa mL^?1. Osmolality (mOsmol kg^?1) and ionic contents (mM L^?1) of the seminal plasma were 274.5±9.0, 70.0±3.4 Na⁺, 28.8±0.9 K⁺, 101.7±3.1 Cl^?, 0.9±0.1 Mg²⁺ and 2.1±0.1 Ca²⁺ respectively. Total protein and glucose were 5.3±0.2 g L^?1 and 76.7±4.3 mM L^?1 respectively. Sperm motility was initiated in a hypo‐osmotic condition, composed of either an ionic (KCl or NaCl) or a non‐ionic (sucrose) activation medium. Duration of sperm motility was very short: <2 min after activation in distilled water. Percentage of motile spermatozoa was significantly higher in an activation medium containing NaCl compared with that of distilled water. An activating medium containing NaCl or KCl higher than 150 mM or sucrose higher than 275 mM totally inhibited the activation of sperm motility. Immediately after sperm activation, wave(s) propagated along the flagellum, but waves were restricted to the proximal part of the flagellum (close to the head) at 1 min post activation. Studied characteristics in the present study were compared with those of other cyprinids for understanding inter‐species differences.     

Differential regulation of pro- and antiapoptotic proteins in fish adipocytes during hypoxic conditions

Worldwide, the frequencies and magnitudes of hypoxic events in estuarine waters have increased considerably over the past two decades. Fish populations are suitable indicators for the assessment of quality of aquatic ecosystems and often comprise a variety of adaptation systems by triggering oxidants, antioxidants and hypoxia-responsive signaling proteins. Signaling pathway may lead to cell survival or cell death which is fine-tuned by both positive and negative factors, which includes hypoxia-inducible factor-1α (HIF1α), heat-shock protein-70 (HSP70), phospho-c-Jun N-terminal kinase 1/2 (p-JNK1/2) and apoptosis signal-regulating kinase-1 (ASK1). In the present study, we attempt to determine stress-mediated signaling changes and molecular mechanism behind the cell survival by comparing adipocytes of fish from field hypoxic condition and laboratory-induced hypoxic condition (in vitro hypoxia). Comparison of field and laboratory studies in fish adipocytes showed differential expression of HIF1α, HSP70, p-JNK1/2 and ASK1 with altered oxidants and antioxidants. Further, the results also suggest that in vitro hypoxic conditions mimic field hypoxic conditions. Trends of hypoxia response were same in in vitro hypoxia of control adipocytes as in Ennore estuary, and hypoxia response was more pronounced in the test adipocytes under in vitro hypoxic condition. Results of the present work suggest that hypoxia is the major crusade of water pollutants affecting fish by differential regulation of pro- and antiapoptotic proteins probably through HSP70. This may play a vital role by providing cytoprotection in pollutant-induced stressed fish adipocytes substantiated by the in vitro hypoxic studies.