黑尾近红鲌精子低温保存方法研究与应用

在4℃条件下,以精子活力为指标,研究了不同浓度的Na~+、K~+、Ca~(2+)、Mg~(2+)、葡萄糖、氨基酸等组成的5种(A、B、C、D、E)精子保存液及其适量添加青霉素对黑尾近红鲌(Ancherythroculter nigrocauda)精子活力的影响。结果显示:1)温度为4℃时,精子活力达80%的保存液A、B、C、D、E的保存时间分别为48、24、60、48、48 h,保存液C的保存时间明显高于其它各组;2)在各保存液中分别添加浓度为2.0×104IU/m L的青霉素,精子活力达80%的保存液A、B、C、D、E的保存时间分别为60、36、156、48、144 h,保存时间延长了12~96 h,保存液C和E的延长时间最长,均为96 h;3)人工授精试验证明,经保存的黑尾近红鲌精子能正常用于人工繁殖,受精率达(90.6±0.8)%~(91.8±0.9)%,与对照组精子受精率(92.4±0.8)%无显著差异。添加青霉素,保存液C的保存效果最好,其次是保存液E。     

pH值、温度、渗透压对鲤鱼精子低温保存效果的影响

就pH值、温度、渗透压等理化因子对鲤鱼精子液态低温保存效果的影响进行了初步研究。结果表明,保存液的pH值为弱酸性(pH6.5)时保存效果最好;低温(0~4℃)条件下,鲤鱼精子的保存效果明显比室温(18~25℃)条件下的保存效果好;在1.2%的NaCl溶液中,鲤鱼精子保存的时间较长。     

超低温冷冻对俄罗斯鲟精子顶体酶活性及DNA损伤的影响

选取6 ind健康的雄性俄罗斯鲟(Acipenser gueldenstaedti),经人工催产后获得成熟的精子,研究超低温冷冻(-196℃)对俄罗斯鲟精子顶体酶活性及DNA损伤影响。结果显示:俄罗斯鲟鲜精中顶体酶的平均活性为(36.18±2.54)μIU·10-6,经过超低温冷冻后,精子顶体酶活性显著降低,添加抗冻保护液精子中顶体酶活性降至(21.55±0.79)μIU·10-6,未添加抗冻保护液精子中顶体酶活性降至(9.58±1.08)μIU·10-6,且三者间有显著性差异(P0.05)。单细胞凝胶电泳结果表明,俄罗斯鲟鲜精彗星率为(37.33±7.77)%,添加抗冻剂后冻精的彗星率为(63.67±5.13)%,未添加抗冻剂直接冷冻彗星率高达(86.00±3.61)%,三者间有显著差异(P0.05)。用CASP分析软件分析测量彗星拖尾长度(L tail)、彗星尾部DNA的相对含量(Tail DNA)、尾动量(TM)、Olive尾动量(OTM)等各项表征DNA损伤的指标,发现冻精组的各项指标均显著高于鲜精组,未添加抗冻剂直接冷冻组又高于添加抗冻剂组,3组间有显著性差异(P0.05)。本研究结果表明:超低温冷冻能导致精子顶体酶活性下降和DNA损伤,抗冻剂对精子具有保护作用。     

虾夷扇贝精子的主要生理特性与低温保存

试验结果表明,虾夷扇贝精子的最适盐度为23.2～32,最适pH 7.0～8.5.4℃,未稀释精液样品保存5 d后,精子仍有(26±4)%的活力,而经海水稀释后的精液保存70 h后,最高活力仅为(11±1.41)%,用抗冻液稀释的精液,4℃保存5 h后,得到最高的精子活力仅为(24.7±4.16)%.添加抗冻剂后,-18℃的保存效果明显低于4℃保存的未稀释样品.用4℃保存的未稀释精液与栉孔扇贝卵子进行杂交试验,保存3 d内的精子获得的授精率与对照组差异不显著(P0.05).杂交组的幼虫可以正常发育至D形幼虫,与自交组无区别.     

大菱鲆精子低温短期保存

大菱鲆(Scophthalmusmaximus)精子仅能体外存活10min左右,根本来不及从养殖场带回实验室,而生产单位的实验条件又有限,这严重限制了大菱鲆精子的实验研究。本文利用TS19做稀释液,6%DMSO作抗冻剂,在4℃下保存大菱鲆精子,每隔一段时间观察1次被保存精子的成活率。结果发现,大菱鲆精子体外存活时间可延长到3h。用保存3h的大菱鲆精子做授精实验,在各种精卵比的情况下,受精率与鲜精对照组没有显著差异。本实验保存大菱鲆精子的方法完全满足了大菱鲆精子短距离运输的要求。     

鲤、鲢、鳙精子低温短期保存研究

本文对鲤(Cygrinus carpio)、鲢(Hypophthalmichthys molitrix)和鳙(Aristichthys nobilis)精液在2—4℃的短期保存进行了实验研究。筛选出了几种较为理想的稀释保护液配方;确定了精液与稀释液的适宜稀释比例为1:1;研究了抗冻剂二甲亚砜(DMSO)对低温保存条件下精于活力的影响,确定了DMSO的适宜添加浓度为6％左右。鲤精在2—4℃保存10天,活力仍高达70％,少数精子存活时间长达12天;鲢和鳙精子在2—4℃保存7天后活力仍高达60％,达到了生产应用的水平。为水产养殖和鱼类育种研究提供了一种简便易行的精液短期保存技术。     

不同温度下精子保存液对黄颡鱼精子活力的影响

以精子的活力为指标,对6种精子保存液在4℃和20~22℃状态下保存黄颡鱼精子的效果进行了比较研究。结果表明:添加了葡萄糖的F保存液效果最好,精子的活力和寿命保持时间最长;在4℃条件下,120h内可保持80%以上活力,寿命持续168h。在黄颡鱼人工繁殖中应用了5种精子保存液进行人工授精,其中4种保存液可替代0.6%生理盐水。     

马氏珠母贝精子的超低温保存

通过比较不同pH值（5．5-9．5）、不同盐度的海水等作为基础液对马氏珠母贝精子保存的影响，选择其中无激活精子作用又对其生理特性（活力，寿命，受精能力等）无影响者，加入二甲亚砜抗冻剂配制成超低温保存的抗冻保护液，精液与保护液按1：10和1：20的比例混合，样品按4组不同的降温程序平衡后转入液氮冷冻，比较其超低温冻存的效果。结果表明：以海水（pH7．5—8．0）为基础液，配制10％DMSO作为抗冻保护液，精液与保护液比例为1：20，在低温（0-4℃）中平衡约30min，在距液氮面15cm、5cm处分别停留5min、10min后转入液氮（-196℃），冻存精子效果良好。冷冻24h、48h及5个月后，在38—40℃下水浴解冻复苏后，用终浓度0．25‰的氨海水刺激，精子存活率可超过60％，受精率可达80％。     

人工养殖西伯利亚鲟精子超低温冷冻保存研究

研究了人工养殖西伯利亚鲟精子的生物学特征及超低温冷冻保存方法。西伯利亚鲟的产精量为113.67±39.86 ml,精子密度为(6.49±3.10)×108/ml,精子活力为(85.4±9.5)%,精子寿命为353±23 s。精子密度与精子快速运动时间、精子寿命之间均存在线性相关,用方程分别表示为:y=1.0384x+1.5089(R2=0.7325);y=2.9069x+74.289(R2=0.6967)。结果表明精子密度可作为一项精子质量评价的标准。通过比较西伯利亚鲟精子在不同稀释液、不同抗冻剂和抗冻剂浓度、降温速率、解冻温度下的保存效果,结果表明:配方2作为稀释液,18%甲醇作为抗冻剂,二步法超低温(-196℃)冷冻保存精子,40℃水浴解冻取得最好的冻后活力,解冻后活力为(51.8±5.8)%。西伯利亚鲟授精的最佳精卵比为106∶1。在此精卵比下用冻精授精分别得到了(72.3±3)%的受精率和(52.9±4.1)%的孵化率,其中受精率与鲜精没有显著性差异,孵化率与鲜精有显著差异(P<0.05)。     

南方鲇精子保存方法的初步研究

以精子被激活后的活力为指标，对6种精子保存液保存南方鲇的效果进行了评估，并对室温（18－20℃）和低温（0－4℃)状态下保存南方鲇精子的效果进行了比较研究。结果表明，在6种精子保存液中，以添加牛血蛋白和氨基酸等营养物质的F精子保存液效果最好，精子的活力和寿命保持时间最长，6d内可保存80％以上活力，寿命持续12.5d。低温下保存效果绝对优于室温下保存。     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of cortisol on hepatic carbohydrate metabolism and responsiveness to hormones in the sea raven,Hemitripterus americanus

The sea raven, Hemitripterus americanus, is a sit-and-wait, low metabolic rate, marine teleost. The objective of this study was to determine i) whether cortisol implantation (50 mg. kg^-1) for 7 days altered hepatocyte metabolism, and hepatocyte responsiveness to epinephrine, glucagon and insulin, and ii) whether 8 weeks of food-deprivation modified the above response. Cortisol implantation significantly increased hepatocyte total glucose production and oxidation from alanine compared to the sham group. There was no cortisol effect on glycogen breakdown, suggesting that the activation of other pathways, including gluconeogenesis, are required to account for the increased glucose production. Epinephrine-mediated (10^-5M) glycogen breakdown and insulin-mediated (10^-8M) total glucose production were enhanced in hepatocytes of cortisol implanted sea ravens, but there were no change in any glucagon (10^-7M) effects. The enhanced glycogen breakdown in the absence of similar increases in total glucose production with epinephrine indicates mobilization of carbohydrate reserves for endogenous use by the liver. Food-deprivation for 8 weeks significantly decreased condition factor, plasma cortisol concentration and liver glycogen content in the sea raven, but had no effect on plasma glucose concentration. Hepatocyte total glucose production and flux rates from alanine increased significantly with food-deprivation. Moreover, food-deprivation increased responsiveness of total hepatocyte glucose production to the actions of glucagon and insulin, but not to epinephrine; none of these effects were modified by cortisol implantation. Our results indicate that cortisol in the sea raven exerts both a direct and an indirect or permissive effect on hepatocyte metabolism by modifying hepatocyte responsiveness to epinephrine and insulin stimulation. Cortisol implantation did not modify the effects of glucagon or food-deprivation in this species.     

Simulation and quantification of enrichment and retention processes in the southern Benguela upwelling ecosystem

Important environmental processes for the survival and recruitment of early life stages of pelagic fishes have been synthesized through Bakun's fundamental triad as enrichment, concentration and retention processes (A. Bakun, 1996, Patterns in the Ocean. Ocean Processes and Marine Population Dynamics. San Diego, CA, USA: University of California Sea Grant). This conceptual framework states that from favourable spawning habitats, eggs and larvae would be transported to and/or retained in places where food originating from enrichment areas would be concentrated. We propose a method for quantifying two of the triad processes, enrichment and retention, based on the Lagrangian tracking of particles transported within water velocity fields generated by a three‐dimensional hydrodynamic model. We apply this method to the southern Benguela upwelling ecosystem, constructing putative maps of enrichment and retention. We comment on these maps regarding main features of the circulation in the region, and investigate seasonal variability of the processes. We finally discuss the results in relation to available knowledge on the reproductive strategies of two pelagic clupeoid species abundant in the southern Benguela, anchovy (Engraulis encrasicolus) and sardine (Sardinops sagax). Our approach is intended to be sufficiently generic so as to allow its application to other upwelling systems.     

Effects of capture,loading density and transport stress on the mortality,physiological responses,bacterial density and growth of rohu <Emphasis Type="Italic">Labeo</Emphasis><Emphasis Type="Italic"> rohita</Emphasis> fingerlings

This study evaluated the effects of three levels of loading density (200, 300 and 400 g L⁻¹) and four levels of duration (1, 3, 6 and 9 h) on the mortality, plasma cortisol and chloride levels and growth of rohu Labeo rohita fingerlings in a transport simulation. While total immediate mortality was low (4–12%), delayed mortality was high (27–49%). Initial capture and handling were found to constitute the most stressful phase as indicated by elevated plasma cortisol. The levels of cortisol continued to rise at 6 and 9 h after transport, with the highest level observed at 9 h after transport. The level of plasma chloride fell after transport across all density levels and treatment durations. Specific growth rate (SGR, %/day) and total bacterial density (cfu/g) were not different between and within treatments, and between treatments and the controls. This study demonstrates that transport has a profound effect on the stress responses of rohu fingerlings. Although the level of immediate mortality was low, the higher level of delayed mortality has serious implications in terms of production inefficiency for grow-out farmers.     

The effects of introduced tilapias on native biodiversity

• 1. The common name ‘tilapia’ refers to a group of tropical freshwater fish in the family Cichlidae (Oreochromis, Tilapia, and Sarotherodon spp.) that are indigenous to Africa and the southwestern Middle East. Since the 1930s, tilapias have been intentionally dispersed worldwide for the biological control of aquatic weeds and insects, as baitfish for certain capture fisheries, for aquaria, and as a food fish. They have most recently been promoted as an important source of protein that could provide food security for developing countries without the environmental problems associated with terrestrial agriculture. In addition, market demand for tilapia in developed countries such as the United States is growing rapidly.
• 2. Tilapias are well‐suited to aquaculture because they are highly prolific and tolerant to a range of environmental conditions. They have come to be known as the ‘aquatic chicken’ because of their potential as an affordable, high‐yield source of protein that can be easily raised in a range of environments — from subsistence or ‘backyard’ units to intensive fish hatcheries. In some countries, particularly in Asia, nearly all of the introduced tilapias produced are consumed domestically; tilapias have contributed to basic food security for such societies.
• 3. This review indicates that tilapia species are highly invasive and exist under feral conditions in every nation in which they have been cultured or introduced. Thus, the authors have concluded that, despite potential or observed benefits to human society, tilapia aquaculture and open‐water introductions cannot continue unchecked without further exacerbating damage to native fish species and biodiversity. Recommendations include restricting tilapia culture to carefully managed, contained ponds, although exclusion is preferred when it is feasible. Research into culture of indigenous species is also recommended.

Copyright © 2005 John Wiley & Sons, Ltd.