分光光度法测定两种蟹类精子密度的研究

蟹类精子计数对其冷冻保存及种群生殖能力的探究有重要意义。为了提高计数蟹类精子数量的速度,建立了分光光度法测定蟹类精子密度的方法。比较了两种蟹类在不同波长（380nm、520nm、760nm）下吸光度（A）与精子密度（C）的关系。结果表明,在520nm波段下三疣梭子蟹（Portunus trituberculatus）精子密度和吸光度之间也存在线性回归关系,其回归方程是：A=0.058×C（R2=0.9928）;中华绒螯蟹（Eriocheir sinensis）精子吸光度与精子密度呈线性回归关系,其回归方程是：A=0.061×C（R2=0.9892）。统计检验表明,使用该方法测定蟹类精子数目,是一种快速而准确的方法,可以加速蟹类精子计数速度。     

不同精子密度冷冻猪5mL细管精液效果比较

采集上海白猪精液进行5mL细管冷冻,比较冷冻密度分别为0.5×109个/mL、0.7×109个/mL、1.0×109个/mL的冻后精子质量。结果表明:3种密度冷冻猪精子,解冻后精子质量差异不显著(P﹥0.05)。     

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)。     

酵母的不同处理方法对其分散度和轮虫培养效果的影响

试验采用多种方式处理酵母,检测处理后的酵母的分散度。处理方式包括搅拌、活化、搅拌+活化。本试验选取30 min和120 min两种活化时间及20℃、30℃、40℃和50℃四个不同的温度梯度对酵母进行处理。结果表明:酵母细胞的分散度为活化后搅拌(直接加水搅拌(直接活化(搅拌后活化。选取酵母细胞密度为2.3×106个/mL,3.3×106个/mL,4.2×106个/mL,5.5×106个/mL,6.4×106个/mL的酵母溶液分别对轮虫进行投喂。结果表明:扩大酵母的分散度,可显著地增加轮虫的增殖率。     

道观河水库的细菌数量及其与环境因子的关系

2000年对道观河水库水生细菌进行6次采样,水生细菌总数量为3.2×103cells/mL,变动范围是3.3×102～9.0×103cells/mL。其中1月数量最大,7月最小;水生细菌的数量随着水体中TN、TP浓度的增加而增加;随浮游植物密度的上升而下降;细菌数量与浮游动物密度也呈现出相反的变化趋势,但相关关系并不显著(P>0.05)。细菌数量表明道观河水库已经受到了中度的污染。讨论了水生细菌的生物调控手段。     

中华倒刺鲃、白甲鱼和岩原鲤精子的生理特性比较

对中华倒刺鲃(Spinibarbussinensis)、白甲鱼(Onychostomasimus)和岩原鲤(Procyprisrabaudi)3种鱼的鲜精在不同水溶液和不同浓度梯度的NaCl溶液中的活力以及精子形态、密度和精液pH值、浓度等进行了比较研究。结果表明:3种鱼的精液pH值都偏弱碱性,位于7·2~7·7之间;精液浓度依次为71·1%,76·1%和71%;精子头长依次为(3·89±0·53)μm,(2·98±0·08)μm和(6·42±0·59)μm,全长依次为(41·63±3·66)μm,(65·67±2·97)μm和(58·89±5·25)μm;精子密度依次为1·527×1010尾/mL,1·336×1010尾/mL和1·362×1010尾/mL。分析表明,3种鱼的精子大小与密度无相关性。在不同水溶液中,3种鱼的精子活力均以池塘水中最高;在不同浓度NaCl溶液中,3种鱼精子的最适浓度位于0·45%~0·55%之间,有效运动时间以中华倒刺鲃最高,为(85·23±12·02)s,其次是岩原鲤,为(50·45±6·89)s,白甲鱼最短,为(31·44±5·53)s。3种鱼的精子全长与精子活力存在负相关性,即精子越长,活力越低,精子越短,活力越高。     

微小亚历山大藻对近江牡蛎清滤率以及麻痹性贝类毒素(PSP)蓄积的影响

为获知牡蛎对有毒微小亚历山大藻(A. minutum)的滤食以及蓄积PSP毒素的规律性,本实验采用细胞毒性为93.42±2.55×10-6 MU/cell的A. minutum藻投喂牡蛎,以无毒扁藻投喂作对照,首先研究了0.5-3.0×103 cells/mL6个不同浓度的毒藻对牡蛎滤食的影响,然后投喂浓度为1.5×103 cells/mL的毒藻,分别研究15 h短期蓄积和5天长期蓄积对牡蛎体内毒素的影响及规律。结果表明,在0.5~1.5×103 cells/mL毒藻浓度范围内牡蛎清滤率较高,超过1.5×103 cells/mL时牡蛎清滤率显著下降(P<0.01),由此确定后期投喂毒藻的浓度“阀值”为1.5×103 cells/mL;短期蓄积实验牡蛎在0~6 h内将有毒藻滤食尽,其体内PSP毒素在6~12 h间达到最大值149.6±10.5 MU/100 g,随后开始下降。长期蓄积实验过程中,牡蛎的清滤率没有发生显著变化(P>0.01),在蓄积实验的第2天牡蛎体内PSP毒素水平超过国家限量标准(400 MU/100g),在5天实验结束时,牡蛎体内PSP毒素水平高达3069.2±178.2 MU/100 g,高于国家限量标准的7.7倍。实验结果作为PSP在牡蛎中蓄积及代谢的重要基础数据,为研究PSP的脱除及净化提供思路。     

Zn2+浓度和藻密度对多刺裸腹溞生命表统计学参数的影响

应用生命表统计学方法,在0.5×10~6、1.0×10~6和2.0×10~6 cells/mL的斜生栅藻(Scenedesmus obliquus)密度下研究了Zn~(2+)浓度(20.0、50.0、80.0、110.0和140.0μg/L)对多刺裸腹溞(Moina macrocopa)生命表统计学参数的影响。结果表明,Zn~(2+)浓度对多刺裸腹溞的种群内禀增长率有显著影响(P0.05),藻密度对多刺裸腹溞的世代时间、净生殖率和种群内禀增长率均有显著影响(P0.05),藻密度和Zn~(2+)浓度间的交相互作作用对多刺裸腹溞各主要生命表统计学参数都没有显著的影响(P0.05)。三食物密度间,多刺裸腹溞的世代时间在1.0×10~6 cells/mL食物密度下显著短于0.5×10~6和2.0×10~6 cells/mL食物密度下;净生殖率和种群内禀增长率均在1.0×10~6 cells/mL食物密度下最高,2.0×106 cells/mL食物密度下最低。50.0和80.0μg/L的Zn~(2+)使多刺裸腹溞的种群内禀增长率显著高于对照组和其它各Zn~(2+)浓度组。Zn~(2+)浓度与多刺裸腹溞的生命表统计学参数中的生命期望和平均寿命在0.5×10~6 cells/mL食物密度下存在着显著的剂量-效应关系,可分别描述为:e0=-0.004x~2+0.063x+348.604(R~2=0.434, P0.05)和t=-0.025x~2-0.507x+84.468(R~2=0.434, P0.05)。     

链状亚历山大藻的培养及麻痹性贝类毒素的提取和检测

为了研究麻痹性贝类毒素(paralytic shellfish poisoning toxins,PSP)的来源和检测,对实验室分离培养的产麻痹性贝类毒素的链状亚历山大藻进行了研究。首先通过接种对数生长期藻细胞,培养8 d内就能达到最高细胞密度(2.4×10⁴ cells/mL)左右,再以0.05 mol/L的醋酸超声波破碎藻细胞提取粗毒素,结合小白鼠生物检测法,经高效液相色谱分析,本株藻主要含有C1/2、GTX4、GTX5和NEO,浓度分别为GTX5(0.827 5)>GTX4(0.339 2)>C2(0.252 6)>NEO(0.126 6)>C1(0.045 5)(单位:μmol/L),毒素粗提液经过Sephadex-G15凝胶层析柱处理,用小白鼠生物法和荧光分光光度法联合检测定位,收集到纯度较高的PSP,本论文工作为将来贝类毒素的研究提供资料。     

3种微藻对海水集约化对虾养殖尾水氮磷的去除效果

基于对虾生物絮团集约化养殖尾水含有高浓度硝态氮和磷酸盐的特征，比较分析钝顶螺旋藻（Spirulina platensis）、牟氏角毛藻（Chaetoceros muelleri）、盐藻（Dunaliella sp.）3种微藻在配制尾水中的存活生长状况及其对无机氮磷的去除效果，以期筛选出适宜的微藻用于后续尾水净化技术。采用显微镜计数法测定藻细胞密度，国标法测定总无机氮、氨氮、硝态氮、亚硝态氮和磷酸盐的含量。结果显示，钝顶螺旋藻在实验前后的藻细胞密度变化不大（P>0.05），约为3.32×106 个/mL和5.88×106 个/mL；牟氏角毛藻和盐藻细胞密度有明显增加（P<0.05），分别从初始的4.00×104 个/mL和2.50×105 个/mL升高至实验结束时的1.66×106 个/mL和1.06×107 个/mL。经过16 d实验，钝顶螺旋藻组对硝态氮和总无机氮去除率分别为79.60%和46.06%，显著高于其他各组（P<0.05），第8天时对磷酸盐的去除率可高达98.55%；牟氏角毛藻组16 d的磷酸盐去除率为98.25%，显著高于其他各组（P<0.05）。研究表明，3种微藻均可在对虾养殖尾水环境中存活，且对尾水氮磷具有较好的净化效果。     

Standardization of photometric measurement of sperm concentration from diploid and tetraploid Pacific oysters, Crassostrea gigas (Thunberg)

To provide necessary standardization of procedures for cryopreservation of sperm, a spectrophotomeric method was developed to determine the sperm concentration of diploid and tetraploid Pacific oysters, Crassostrea gigas. Wavelengths of 380, 550, 581 and 780 nm were compared, and 550 and 581 nm were found to be the most sensitive and reliable. A linear relationship between sperm concentration and photometric absorbance was observed for sperm concentrations between 2 × 10⁷ and 2 × 10⁹ cells mL^?1. The regression equation for the standard curve at 550 nm for sperm of diploid oysters was Y=?8.528+1.165 log X. The equation for sperm of tetraploid oysters was Y=?8.844+1.236 log X. The equation at 581 nm for sperm of diploid oysters was Y=?8.07+1.104 log X. The equation at 581 nm for sperm of tetraploid oysters was Y=?8.331+1.167 log X. Comparisons derived from the standard curves at 581 nm between observed values and the predicted values indicated good agreement for sperm from diploid (coefficient of determination, r²=0.983) and tetraploid (r²=0.980) oysters.     

Development of a spectrophotometric technique for sperm quantification in the spermcasting Australian flat oyster Ostrea angasi Sowerby

Sperm quantification is vitally important when sperm concentration is required for standardization of different fertilization treatments in a hatchery. Although the haemocytometer method is generally used to determine sperm concentration, the procedure is tedious and the attributes are not suitable for handling a large number of sperm samples within a short period. In this study, the efficiency of sperm concentration determination was improved in the spermcasting oyster Ostrea angasi Sowerby by optimizing the regression model and parameters critical to spectrophotometric reading. Although sperm concentration can be estimated in a wide range of wavelengths, the 350‐nm wavelength produced the best fit to the regression model (y = 1 × 10^?8 x + 0.163; r² = 0.996). In addition, the sperm counts estimated with this model were similar to the haemocytometer counts. The reading repeatability of this technique was further validated with samples from different individuals. Comparisons with literature suggest that when the spectrophotometric technique is applied to a new species for estimating sperm concentration, the regression relationship between sperm concentration and wavelength reading should be reassessed due to species‐specific discrepancy.     

Estimation of sperm concentration of wild and reconditioned brown trout, Salmo trutta L.

Sperm concentrations were assessed for milt samples taken from three stocks of brown trout, Salmo trutta L., (wild resident trout, and reconditioned and seareared sea trout) using direct counts, spermatocrit and an optical density of 1:1000 dilutions. There were significant linear relationships between spermatocrit and absorbance, and both spermatocrit and absorbance with sperm concentration. The results gave sperm concentrations from 2.2 × 10⁹ mL^?1 to 26 × 10⁹ mL^?1. Mean concentrations differed significantly between stocks; the reconditioned males had a mean concentration of 9.1 × 10⁹ mL^?1, sea-reared males 18.0 × 10⁹ mL^?1 and 8.0 × 10⁹ mL^?1 in 1994 and 1995, respectively, and wild resident males 13.3 × 10⁹ mL^?1. Sperm concentrations from all three stocks of trout were negatively correlated with length, and therefore, age. It was indicated that sufficient milt was being produced to achieve good rates of fertilization, although some of the larger trout did have low sperm counts, suggesting that fertilizing ability may decrease with male size or age within any one stock.     

Induced spermiation in 3-year-old sterlet, Acipenser ruthenus L.

Spermiation in 3‐year‐old sterlet, Acipenser ruthenus (L.), males maintained under warm water conditions was induced by intramuscular injection of either (i) (D‐Ala⁶)‐GnRH‐ProNHEt (Kobarelin); (ii) mammalian GnRH analogue+metoclopramide (Ovopel); or (iii) human chorionic gonadotropin (Biogonadyl). The volume of milt, sperm concentration and motility were measured. A higher percentage of spermiating males was obtained after Kobarelin or Ovopel treatment (81.8% and 77.7% respectively) in comparison with fish treated with Biogonadyl (40.0%). However, only stimulation with Ovopel guaranteed motile spermatozoa in all spermiating males. Moreover, treatment with Ovopel resulted in the highest average milt volume, sperm concentration and motility. The average total number of motile spermatozoa (milt volume×sperm concentration×sperm motility) per individual was 0.99×10⁹, 5.31×10⁹ and 0.02×10⁹ after stimulation with Kobarelin, Ovopel or Biogonadyl respectively. Our data indicate that Ovopel is a good stimulator of spermation in sturgeons. Moreover, the time of sexual maturation could be significantly reduced in sturgeons maintained in cages under warm water conditions.     

Spermiation of paddlefish (Polyodon spathula,Acipenseriformes) stimulated with injection of LHRH analogue and carp pituitary powder

The potential of carp pituitary powder (CPP) at one dose, or the luteinizing hormone-releasing hormone (LH-RH) analogue, des–Gly¹⁰,(d–Ala⁶)–LH-RH–ethylamide, at three different doses to stimulate spermiation in paddlefish (Polyodon spathula) was tested. Single injections of the LH-RH analogue at 0.2, 0.1, or 0.05 mg·kg^–1 increased the number of spermatozoa per kilogram of body weight (kg^–1 b.w.) by 4.7, 3.4, and 3.4 times respectively compared to control, but the number of spermatozoa per kilogram of body weight decreased with CPP (4 mg·kg^–1) by 1.7 times compared to the control. The LH-RH analogue prolonged active spermiation, with numbers of spermatozoa ranging from 7.69 to 1.19 × 10⁹ kg^–1 b.w. up to 96 h after treatment. Analysis of variance showed significant influence of experimental groups on volume of sperm per male and per kilogram of body weight, and the total number of spermatozoa per kilogram of body weight, but insignificant influence on the total number of spermatozoa per male. The percentage of motile spermatozoa was not different between experimental groups for sperm collection at different times after injection. A very high positive correlation (r = 0.93) was obtained between sperm concentration and sperm transmittance measured with a spectrophotometer. This relationship was described with the following linear regression: sperm concentration (× 10⁹ mL^–1) = 1.3244 X^–0.9969, where X is the percentage of sperm transmittance.     

The parameters of energetic status of the wild Atlantic salmon (Salmo salar L.) spermatozoa

The energetic status of fresh sperm of wild Atlantic salmon (Salmo salar L.) was presented in selected males (n=10) with motility rate ≥80%. Purine and pyridine nucleotides: adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, inosine monophosphate, nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; nucleosides: adenosine, guanosine and inosine; bases: hypoxanthine, xanthine and uric acid were determined using the high‐performance liquid chromatography method. The average value of adenylate energy charge in the group was 0.77 ± 0.10, and the mean total adenine nucleotide content (TAN) was 65.1 ± 18.3 pmol × 10^?6 spermatozoa. The mean concentrations of ATP, ADP and AMP were 43.5, 11.8 and 9.9 pmol × 10^?6 spermatozoa respectively. The concentrations of the other energetic nucleotides studied were lower. In all males studied (n=23) with a motility rate from 0% to 95%, no statistically significant correlation between the per cent of motile sperm and ATP concentration was found (R_s=0.35), whereas the correlation between the per cent of motile sperm and ADP was statistically significant (R_s=0.60). A negative correlation was found between hypoxanthine and the per cent of motile spermatozoa (R_s=?0.44). Our results suggest that hypoxanthine is the final product of salmon spermatozoa of adenine nucleotide catabolism.     

Optimization of sperm irradiation protocol for induced gynogenesis in Siberian sturgeon,Acipenser baerii

The great diversity of optimal UV irradiation doses are used for DNA inactivation in fish sperm forcing authors to repeat optimization of irradiation treatment every time. Analysis of sperm UV irradiation protocol for induction of gynogenesis showed the importance of sperm UV light absorption estimations. The UV absorption investigation in Siberian sturgeon sperm showed average extinction coefficient 7.69 × 10^?8 ± 1.04 × 10^?8 cm². It is resulted in high heterogeneity of UV irradiation of undiluted sperm samples. Therefore, it is strongly suggested to specify doses only with defined concentration of spermatozoa; otherwise, the difference in absorbance level between samples can bring a significant error to optimal UV dose estimation. This was confirmed by UV-irradiated sperm motility investigation. Results of motility investigation of UV-irradiated sperm revealed high sensitivity of Siberian sturgeon spermatozoa motion mechanisms to UV irradiation, with complete loss of motility after homogeneous UV irradiation at doses above 2,000 J/m². Partial gynogenesis was conducted using diluted and undiluted sperm. Ploidy level of hatched larvae was estimated by flow cytometry. Percentage of haploid hatched larvae revealed sperm DNA inactivation efficiency. The highest percentage of haploid putative gynogenotes 19.67 ± 4.19 % was obtained at UV irradiation dose 200 J/m² with sperm diluted to 1:4.