Characterization and Inhibition of Nitrite Uptake in Shortnose Sturgeon Fingerlings

Abstract

Efforts are underway to culture the endangered shortnose sturgeon Acipenser brevirostrum for possible reintroduction. As part of a larger project to develop culture techniques for this species, the uptake of nitrite was evaluated in fingerlings (16.5 ± 4.85 g; mean ± SD). Plasma nitrite concentrations increased significantly with exposure time (0–5 d) and dose (0–4 mg nitrite-N/L). Shortnose sturgeon fingerlings were able to concentrate nitrite in their plasma to more than 63 times the environmental concentration. Chloride, as either sodium chloride or calcium chloride, partially inhibited nitrite uptake. However, calcium chloride was a better inhibitor. After previous exposure (2 d at 2.13 ± 0.080 mg nitrite-N/L) plasma nitrite-N decreased from 165.5 to 36.7 mg/L during a 3-d simultaneous exposure to 2.13 ± 0.080 mg nitrite-N/L and treatment with 40 mg chloride/L as calcium chloride. The addition of calcium chloride to the water appeared to be an effective means of preventing nitrite uptake and treating nitrite toxicity in hatchery-reared shortnose sturgeon fingerlings.     

Responses of Blue Catfish and Channel Catfish to Environmental Nitrite

Abstract

In nitrite-exposure experiments, percent methemoglobin, plasma nitrite concentration, and plasma chloride ion concentration were compared between channel catfish Ictalurus punctatus and blue catfish I. furcatus exposed to sublethal levels of nitrite for 48 h at 25°C. In nitrite-recovery experiments, fish exposed to elevated environmental nitrite for 12 h were transferred to freshwater, and blood characteristics were monitored during the 24-h recovery period. Blue catfish appeared to be more resistant to environmental nitrite than channel catfish. Methemoglobin levels (percent of hemoglobin in methemoglobin form) were significantly lower in blue catfish than in channel catfish. Maximum plasma nitrite concentrations were 137 mg NO₂-/L plasma in blue catfish and 164 mg NO₂-/L plasma in channel catfish. Percent methemoglobin and plasma nitrite concentration were closely correlated. Plasma chloride decreased initially with exposure to nitrite but quickly returned to control levels. Blue catfish exposed to nitrite at 10°C required 1 week to recover when placed in nitrite-free water. The methemoglobin reductase enzyme apparently functioned at a slow rate in fish acclimated to cold temperatures.     

The Effect of High Total Ammonia Concentration on the Survival of Channel Catfish Experimentally Infected with Flavobacterium columnare

Abstract

Ammonia concentrations in water can affect the severity of Flavobacterium columnare infections in fish. Two trials lasting 7 d each were conducted to determine the effect of a single immersion flush treatment of total ammonia nitrogen (TAN; 15 mg/L) on the survival of channel catfish Ictalurus punctatus infected with F. columnare; the chemical was added while the water flowed continuously through the tanks. Both trials consisted of four treatments: (1) no ammonia exposure and no bacterial challenge (control), (2) ammonia exposure only, (3) bacterial challenge only, and (4) both ammonia exposure and bacterial challenge. Two hours after exposure to ammonia, the highest un-ionized ammonia level was 0.43 mg/L. The percent un-ionized ammonia is based on TAN, temperature, and pH. Caudal fins from three fish in each treatment were sampled at 24 h posttreatment to be analyzed by quantitative real-time polymerase chain reaction (qPCR). No significant difference in survival (mean ± SE) was noted between the channel catfish in treatment 1 (95.2 ± 1.2%) and those in treatment 2 (95.6 ± 1.0%); however, survival in both treatments 1 and 2 differed significantly from that in treatments 3 (8.5 ± 4.5%) and 4 (41.8 ± 12.7%). Treatment 4 catfish had significantly higher survival than treatment 3 catfish. Quantitative PCR data showed that treatment 4 fish had significantly less F. columnare (7.6 × 10⁵) than did treatment 3 fish (1.2 × 10⁷), and treatment 2 fish (8.5 × 10³) had significantly less bacteria than did treatment 1 fish (6.9 × 10⁴), indicating that ammonia limited the F. columnare infection. The highest mean concentration of the bacteria (3.9 × 10⁷) was found on moribund fish. The ammonia concentrations tested did not negatively influence fish survival but interfered with the infection process. An in vitro assay was also conducted to evaluate the direct effects of ammonia on F. columnare.

Received September 15, 2010; accepted May 7, 2011     

Digestive enzymes along digestive tract of a carnivorous fish Glyptosternum maculatum (Sisoridae,Siluriformes)

This study attempted to determine the presence, distribution and levels of digestive proteases, pepsin, amylase and lipase in the homogenates of digestive tract of a freshwater, cold‐adapted, carnivorous teleost of Tibet, Glyptosternum maculatum (n = 10, mean weight: 129.6 ± 28.9 g, mean length: 195.1 ± 16.6 mm, approximately 6 years old), at different temperatures and pH levels. The descending order of protease activity was as follows: stomach (pepsin, 16.16 ± 0.96 U/mg protein), anterior intestine (3.18 ± 0.25 U/mg protein), posterior intestine (1.76 ± 0.21 U/mg protein) and middle intestine (1.52 ± 0.23 U/mg protein). Amylase activity levels in descending order were as follows: anterior intestine (0.0062 ± 0.0007 U/mg protein), stomach (0.0032 ± 0.0009 U/mg protein), middle intestine (0.0023 ± 0.0005 U/mg protein) and posterior intestine (0.0023 ± 0.0004 U/mg protein). The highest lipase activity was found in the anterior intestine (0.83 ± 0.25 U/mg protein), followed by posterior intestine (0.51 ± 0.19 U/mg protein), middle intestine (0.48 ± 0.09 U/mg protein) and stomach (0.39 ± 0.10 U/mg protein). The optimal temperature and pH level for protease were 50 °C and 9.0–10.0, respectively, along the intestine. The pepsin optima in the stomach were 30 °C and 2.0 respectively. The optimal temperature for amylase was found to be 30 °C along the digestive tract and the optimal pH was 7.0 in the intestine and 6.0 in the stomach. The optimal temperature and pH levels of lipase were 30 °C, pH 6.0 for the stomach and 40–50 °C, pH 8.0 for the intestine, respectively. Hepato‐somatic index was 1.35 ± 0.2% and relative intestine length was 0.90 ± 0.19. The weight ratio of the stomach and intestine to body weight was 1.63 ± 0.43% and 1.57 ± 0.24% respectively.     

Effect of Waterborne Potassium Permanganate Exposure on Manganese Content in Liver and Axial Muscle of Channel Catfish

Abstract

Adult channel catfish Ictalurus punctatus were exposed to waterborne potassium permanganate for 12 weeks to determine if such exposure would alter the manganese content of axial muscle or liver tissue. Continuous exposure to 0.5 mg KMnO₄/L or exposure to 1 or 2 mg KMnO₄/L on alternate days did not cause a significant increase in manganese in axial muscle or liver tissue. The mean (±SE) concentration of manganese in axial muscle of unexposed controls was 0.262 ± 0.018 mg/kg (wet weight). Means of manganese concentrations in axial muscle of the three exposure groups during the 12 weeks of exposure were 0.289 ± 0.021 mg/kg, 0.269 ± 0.018 mg/kg, and 0.239 ± 0.013 mg/kg for 0.5 (continuous), 1, or 2 mg/L (alternate days), respectively. At specific sampling times there were differences between controls and exposure groups; however, no trend toward higher or lower manganese concentrations in muscle could be detected within groups. The mean (±SE) concentration of manganese in liver tissue of controls was 1.67 ± 0.09 mg/kg (wet weight). Manganese concentrations in liver tissue of the three exposure groups were 1.57 ± 0.07 mg/kg, 1.68 ± 0.08 mg/kg, and 1.58 ± 0.10 mg/kg, for 0.5 (continuous), 1, or 2 mg/L (alternate days), respectively. Manganese was thought to accumulate in liver tissue, however, there were no statistically significant differences between those groups and the controls. Results suggest that potassium permanganate used as a waterborne disease therapeutant for channel catfish does not alter manganese content of edible muscle of channel catfish and should not present any hazard to human consumers.     

Molluscicidal activity of Carica papaya and Areca catechu against the freshwater snail Lymnaea acuminata

The molluscicidal activity of seed and lyophilized latex powder of Carica papaya and seed powder of Areca catechu against the vector snail Lymnaea acuminata was studied. The toxicity of these plant products was time and dose dependent. The toxicity of C. papaya lyophilized latex powder (LC(50) at 96 h: 8.38 mg/l) was more pronounced than that of A. catechu seed powder (LC(50) at 96 h: 12.32 mg/l) and C. papaya seed powder (LC(50) at 96 h: 61.56 mg/l). Ethanolic extracts of C. papaya seed and A. catechu seed were more toxic than their other extracts. The ethanolic extract of A. catechu seed (LC(50) at 24h: 17.21 mg/l) was more effective than the ethanolic extract of C. papaya seed (LC(50) at 24h: 53.38 mg/l). The LC(50) of column-purified fraction of A. catechu seed at 96 h was 3.99 mg/l, whereas that of C. papaya seed was 7.06 mg/l. C. papaya and A. catechu may be used as potent molluscicides since the concentrations used to kill the snails were not toxic for the fish Colisa fasciatus which shares the same habitat with the snail L. acuminata.     

鸡尿液收集方法及其理化指标和尿沉渣的研究

对 2 0羽 45日龄伊沙蛋雏鸡进行结肠造口术 ,收集 2 4h的尿液及尿沉渣 ,并对鸡尿液的理化指标及尿沉渣进行分析研究。结果显示 :鸡的尿液呈淡黄色 ,并有稠粘液体性质 ,鸡的尿量为 :(37 34± 8 0 0 )mL ,尿液pH值为 6 58± 0 32 ,尿密度为 1 0 1 8± 0 0 0 9,尿酸为 (398 951±51 2 91 ) μmol/L ,钙为 (5 580± 0 2 4 1 )mmol/L ,无机磷为 (1 2 1 7± 0 996)mmol/L ,镁为 (3 735± 0 0 1 0 )mmol/L ,钠为 (1 37 2 50±1 3 789)mmol/L ,钾为 (41 81 0± 1 887)mmol/L。扫描电镜下 ,尿沉渣中球形物较多 ,混有少量的针状晶体 ,表明主要含尿酸盐。能谱分析表明 ,上述结构的主要组成元素为钙、钾及少量的镁和钠     

纳米光催化环境改良剂降低猪舍氨气浓度的研究

试验针对夏季北方地区正常生产的哺乳母猪舍、保育猪舍、育肥猪舍的氨气浓度,检验纳米光催化环境改良剂干粉的处理效果。结果表明,处理20 min后猪舍平均氨气浓度快速降至1.11 mg/L～1.61 mg/L之间。哺乳母猪舍处理后72 h内保持较好的使用效果,96 h恢复至处理前氨气的浓度水平。保育猪舍与育肥猪舍再处理后96 h仍然保持较好的处理效果。     

烟酸铬对西门塔尔牛瘤胃液pH值和氨态氮浓度的影响

试验选用4头平均体重(500±20)kg,年龄4岁,装有永久性瘤胃瘘管的西门塔尔牛阉牛,采用4×4拉丁方设计,以混合精料和风干玉米秸秆为基础日粮,研究日粮添加烟酸铬(0、40、801、20 mg/d)对瘤胃液pH值及氨态氮浓度的影响。结果表明:日粮添加烟酸铬对瘤胃pH值无显著差异(P>0.05);80 mg/d组瘤胃液氨态氮浓度显著低于对照组,其他各组间无明显差异(P>0.05)。综合以上结果,烟酸铬适宜添加水平为80 mg/d。     

Safety of Strontium Chloride as a Skeletal Marking Agent for Pacific Salmon

Abstract

The purpose of this study was to evaluate the biological effects associated with administering strontium chloride as a marking agent to age-0 Chinook Salmon Oncorhynchus tshawytscha fry. Fish were held in a 0× (0 mg/L), 1× (3,000 mg/L; current standard dosage), 3× (9,000 mg/L), or 5× (15,000 mg/L) solution of strontium chloride for 72 h (three times the standard duration of 24 h). The mortality among fish in the 5× strontium chloride exposure group was significantly higher than that observed in the other groups. A dose-related effect on general fish behavior and on feeding behavior was observed. Fish in all test tanks appeared to feed to satiation, except for fish in the 5× tanks during days 2 and 3. Fish in all other test tanks behaved normally. No dose-related effect on fish growth was detected. Histopathological evaluations showed that fish in the 5× exposure group had a significantly higher number of gill lesions than the 0× group. Our mortality, behavioral, and histological assessments suggested that juvenile Chinook Salmon could be safely immersed for three consecutive days in a 9,000-mg/L solution of strontium chloride. This finding potentially expands the present 1,000–3,000-mg/L dosage and 24-h holding period that can be used to mark juvenile fish with strontium chloride solutions. The research also provides necessary target animal safety data for U.S. Food and Drug Administration approval of strontium chloride as an alternative marking method that is suitable for fish with a short holding time.

Received February 19, 2017; accepted July 16, 2017 Published online September 22, 2017     

Blood chemistry values of juvenile red pacu (Piaractus brachypomus)

Abstract: Blood samples were collected from 29 juvenile red pacu ( Piaractus brachypomus ), ornamental freshwater fish, to establish baseline blood chemistry values. Mean (minimum-maximum) values, obtained by automated bichromatic analysis and ion selective electrode analysis, were as follows: sodium, 150.4 (146–159) mmol/L; potassium, 3.93 (2.7–5.0) mmol/L; chloride, 138.7 (128–150) mmol/L; total CO₂, 7.5 (6–10) mmol/L; albumin, 0.86 (0.5–1.0) g/dL; lactate dehydrogenase, 237.8 (65–692) IU/L; aspartate aminotransferase, 49.1 (0–125) IU/L; creatinine, 0.31 (0.2–0.4) mg/dL; calcium, 10.80 (9.5–12.5) mg/dL; anion gap, 6.89 (1.2–12.5) mmol/L; and phosphorus, 7.29 (4.1–8.9) mg/dL.     

饲料维生素C含量对圆斑星鲽幼鱼抗氨氮胁迫能力的影响

氨氮污染是水产养殖中重要的污染物,因此提高鱼体抗污染应激能力至关重要。本试验采用不同维生素C含量的饲料饲喂圆斑星鲽幼鱼,探究饲料维生素C含量对圆斑星鲽幼鱼抗氨氮胁迫能力的影响。试验在(12.5±1.5)℃的水温下进行,选择体重为(38.0±0.8)g的健康圆斑星鲽幼鱼,随机分为7组(每组3个重复,每个重复30尾),分别投喂维生素C含量为10.2(对照)、249.1、402.8、616.2、769.5、909.4和1 177.8 mg/kg的试验饲料8周。投喂试验结束后,从每个重复取10尾鱼,用20 mg/L的氨氮胁迫24 h。结果表明:无论是氨氮胁迫前还是氨氮胁迫后,在饲料维生素C含量达到769.5 mg/kg时,肝脏和肌肉中维生素C积累达到饱和,再继续升高饲料维生素C含量,肝脏、肌肉中维生素C含量不再显著升高(P0.05)。除1 177.8 mg/kg维生素C组外,无论是氨氮胁迫前还是氨氮胁迫后,各维生素C添加组圆斑星鲽幼鱼血清中过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性均显著高于对照组(P0.05)。除616.2 mg/kg维生素C组血清中CAT活性无显著变化(P0.05)外,氨氮胁迫使各组血清中CAT和SOD活性显著降低(P0.05),但添加维生素C可以减少降低的幅度。无论是氨氮胁迫前还是氨氮胁迫后,各维生素C添加组鳃丝Na+/K+-ATP酶活性均显著高于对照组(P0.05)。氨氮胁迫使对照组及909.4和1 177.8 mg/kg维生素C组鳃丝Na+/K+-ATP酶活性显著降低(P0.05),其他组则无显著变化(P0.05)。氨氮胁迫使各组血清中葡萄糖和乳酸含量显著升高(P0.05),对照组血清中皮质醇含量显著升高(P0.05)。此外,氨氮胁迫使各组血清中总铁结合力显著降低(P0.05)。综合各项测定指标,圆斑星鲽幼鱼饲料中维生素C含量在402.8~616.2 mg/kg时可有效提高机体抗氨氮胁迫能力。     

Rates of change in liver copper concentration in cattle given a copper-deficient diet,with or without pre-treatment with tetrathiomolybdate,for evaluation of two parenteral copper supplements

AIM: To minimise the impact of initial variation in liver copper (Cu) on assessments of Cu supplements for cattle in depletion/repletion experiments.

METHODS: Efficacy of two Cu injections was assessed with 18 calves, weighing 200–250 kg, given a Cu-deficient barley diet, containing 4.1 mg Cu/kg dry matter (DM) and added molybdenum (3 mg/kg) and sulphur (3 g/kg). Initial liver biopsy Cu ranged from 3.15–14.17 mmol/kg DM and nine calves with the highest values were given three subcutaneous injections of 235 mg tetrathiomolybdate (TTM) after 42–46 days depletion to lower liver Cu. Untreated (L) and TTM-treated (H) calves were ranked separately for liver Cu after 50 days depletion and allocated to one of three treatments: 100 mg Cu given subcutaneously as CuCaEDTA in either a paraffin (CuP) or aqueous base (CuA) after 56 days depletion (Day 0) or no injection (O). Thereafter, plasma and liver biopsy Cu were measured every 2–4 weeks for 16 weeks. Responses in liver Cu to Cu injections were compared with and without log_e transformation and by linear regression.

RESULTS: Prior to Cu injection, the fractional decline in liver Cu concentration (FDLCu) after 50 days depletion was 0.64 (SE 0.066) and 0.80 (SE 0.090) in H and L calves, respectively (p=0.09) and mean liver Cu did not differ on Day ?6 (6.65 (SE 0.516) and 4.91 (SE 0.681) mmol/kg DM, respectively). Mean plasma Cu was higher in H than L calves on Day 0 (16.6 (SE 0.52) and 13.3 (SE 0.49) μmol/L, respectively (p<0.001)). Rates of decline in log_e liver Cu between Days 0–84 in treatments L and H were: 0.0138 and 0.0071 for Groups O; 0.0033 and 0.0016 for Groups CuP; 0.0073 and 0.0049 for Groups CuA (pooled SE 0.0014) mmol/kg DM/day, respectively. Between Days 84–114, FDLCu was uniformly high across experiments and groups (0.59 (SE 0.042)). Cu injections did not affect plasma Cu, which remained 3.1 (SE 0.41) umol/L higher in Experiment H than in L (p=0.017).

CONCLUSIONS: The use of rates of change in liver copper concentrations improved the assessment of efficacy for two parental copper supplements and that of pre-treatment with tetrathiomolybdate, which, contrary to expectation, slowed Cu turnover by mechanisms that remain unclear.     

Treatment of Ichthyophthiriasis in Rainbow Trout and Common Carp with Common and Alternative Therapeutics

Abstract

The goal of this laboratory study was to provide better knowledge about the treatment of ichthyophthiriasis (causative agent: Ichthyophthirius multifiliis, a ciliate bacteria) in rainbow trout Oncorhynchus mykiss and common carp Cyprinus carpio. The following questions were investigated: (1) the effectiveness of different chemicals (formalin, sodium chloride, hydrogen peroxide, Perotan, Virkon, Aquahumin, Baycox, and Ivomec) and at different concentrations and durations of application, (2) the number of treatments and the time intervals between treatments that were necessary to remove the parasite, and (3) how treatment effectiveness differed between the two species. The most effective treatment was a 37% stock solution of formalin at 110 μL/L of bath water for 1 h in rainbow trout and for 2 h in common carp. Aquahumin (150 μL/L for 2 h) was effective in slightly or moderately infected rainbow trout and at low water temperatures, but it was not effective for common carp. All other tested chemicals were ineffective. With formalin and Aquahumin, five treatments were necessary to remove I. multifiliis infestation. At 10 ± 1°C, the parasites were eradicated when the treatment was performed at 48-h intervals. At 18 ± 1°C the infestation was eliminated when treatment was performed at 24-h intervals but not at 48-h intervals. At 25 ± 1°C, treatment at 24-h intervals was ineffective; however, shorter intervals between treatments might improve treatment efficacy at this temperature. In contrast, the number of treatment repetitions played a minor role, and parasites were eliminated with five treatments in all experiments when the type of chemical and treatment interval were optimal.     

Electrolyte and protein levels in bovine tears

Electrolyte and protein levels were measured in lacrimal fluid collected from both eyes of 20 calves. In the 40 samples analysed mean levels of sodium were 138 ± 1·0 mmol/l, potassium 20 ± 0·5 mmol/l, calcium 0·79 ± 0·04 mmol/l, magnesium 0·56 ± 0·02 mmol/l, chloride 133 ± 1·5 mmol/l, inorganic phosphate 0·18 ± 0·03 mmol and protein 7 ± 0·4 g/l. Tear collection rate was 130 ± 13 μl/min. Significant positive correlations were found between levels of sodium and chloride (0·55, P < 0·01) and calcium and magnesium (0·42, P < 0·01), and negative correlations between sodium and potassium (0·67, P < 0·001), calcium and chloride (0·37, P < 0·05), calcium and tear collection rate (0·32, P < 0·05), and magnesium and tear collection rate (0·32, P < 0·05). Pairing the right and left eyes of each animal, all measured components showed significant correlations except inorganic phosphate, protein and collection rates.     

Dietary manipulation to reduce aerial ammonia concentrations in nursery pig facilities.

Two 4-wk trials (preliminary study) and three 5-wk trials (major study) were conducted to determine the effects of adding Yucca schidigera extract or anhydrous calcium chloride to nursery diets on the growth performance of nursery pigs and aerial ammonia concentration. The pigs were weaned between 13 and 15 d of age and had an initial BW of 3 to 6 kg. In each trial, pigs were allotted to three identical pig nursery rooms that were environmentally regulated. There were three diets (one diet per room): 1) control, containing 23% CP; 2) control plus 125 ppm of Yucca schidigera extract; and 3) control plus 1.95% anhydrous calcium chloride. Growth performance was recorded weekly. Aerial ammonia concentration was measured daily using aspiration detector tubes and during the last week of each trial using diffusion tubes. Manure samples were collected twice a week during the experimental period to determine ammonia and N concentrations and pH. Plasma urea concentration was determined in blood samples collected from the pigs at the end of each trial. Data were analyzed using split-plot and Latin square designs for the preliminary and major studies, respectively. Feed intake was similar among pigs fed all three diets. There were no differences in ADG and ADG/ADFI (G/F) between pigs fed the control diet and pigs fed the yucca extract diet (P > or = 0.41). In all trials, pigs fed the calcium chloride diet had lower ADG and G/F than pigs fed the other two diets (P < 0.05). In the preliminary study, aerial ammonia tended to be greater in the rooms in which pigs were fed the control diet than in the rooms with pigs fed the yucca extract diet (P = 0.08) and the calcium chloride diet (P = 0.11). In the major study, aerial ammonia increased weekly (diet x week; P < 0.001) in all rooms. In the 4th wk, ammonia concentrations were greater (P < 0.001) in the rooms in which pigs were fed the control diet than in the rooms in which the other two diets were fed. Dietary treatment had no effect on plasma urea concentration (P > or = 0.10), manure ammonia and N concentrations (P > or = 0.50), and manure pH (P > or = 0.78). Although aerial ammonia concentrations were relatively low, the addition of Yucca schidigera extract or calcium chloride to the diet of nursery pigs reduced ammonia concentrations in the nursery rooms.     

Toxicity of Copper Sulfate to Flavobacterium psychrophilum and Rainbow Trout Eggs

Abstract

Tests were conducted to determine the concentrations of copper sulfate needed to kill Flavobacterium psychrophilum, the cause of bacterial coldwater disease, either in vitro or on Rainbow Trout Oncorhynchus mykiss eggs. For the in vitro test, a plastic strip dipped in a solution of F. psychrophilum was exposed for 15 min to copper sulfate solutions of 0, 1, 5, 10, 20, 35, 50, 75, or 100 mg/L. Bacteria were “too numerous to count” at concentrations ≤10 mg/L CuSO₄; significant reductions in prevalence relative to untreated controls were noted for concentrations ≥35 mg/L. However, CFUs were still observed at 50 and 75 mg/L (20% of plates with tryptone yeast extract salts media). No yellow-pigmented CFUs typical of F. psychrophilum were observed at 100 mg/L CuSO₄. For the in vivo test, eggs were exposed for 15 min to 100, 300, 500, and 700 mg/L CuSO₄ or 100 mg/L iodine (control). Survival to hatch was significantly lower at 500 (44.3 ± 15.2%, mean ± SD) or 700 mg/L CuSO₄ (1.7 ± 0.8%) than for controls treated with 100 mg/L iodine (93.6 ± 0.9%) or at copper sulfate concentrations ≤300 mg/L. The 15-min LD₅₀ and LD₁₀ for copper sulfate were 461 mg/L (95% confidence interval: 457–466 mg/L) and 259 mg/L (251–266 mg/L). The prevalence of yellow CFUs at 100 mg/L CuSO₄ (40.0%) was significantly higher than in untreated controls. Significant reductions in yellow CFUs were achieved using 300, 500, or 700 mg/L CuSO₄ (7.5, 2.5, or 0.0% of plates with CFUs, respectively) or 100 mg/L iodine (2.5%), relative to untreated control eggs. Overall, since the concentrations of copper sulfate required to eliminate F. psychrophilum were toxic to the eggs, copper sulfate is not recommended for coldwater disease control in Rainbow Trout eggs based on conditions and parameters in this study.

Received July 7, 2011; accepted March 17, 2013     

Germination Ecology of Cenchrus biflorus Roxb.: Effects of Environmental Factors on Seed Germination

Better understanding related to germination and seedling emergence of plant species assists in predicting the potential distribution and provides insight for efficient management. Cenchrus biflorus Roxb. has been considered as potential forage species in arid environment due to its high nutritive values, prolific seed production, and tolerance to extreme temperature and prolonged drought conditions. A series of laboratory and greenhouse assays were conducted to determine the effect of different environmental factors, such as temperature, light, pH, salinity, osmotic potential, and seed burial depth on the germination and seedling emergence of C. biflorus. The maximum germination (95%) was recorded at 35°C/25°C, followed by 40°C/30°C; however, minimum germination was observed at 45°C/35°C (17.5%). Light significantly promoted the germination with maximum percentage (97.5%) when seeds were exposed to altering light and dark conditions (12/12 h). The osmotic potential for the 50% inhibition of C. biflorus germination was –0.4 MPa, although some seed germinated at –0.8 MPa (12.5%). Germination decreased from 97.5% to 12.5% as salinity stress increased from 0 mM to 200 mM sodium chloride (NaCl) with no germination > 200 mM. Seed germination was significantly affected by pH levels and was between 27.5% and 92.5% at 5–8 pH, respectively. No seedling emerged when seeds were placed on the soil surface; maximum seedling emergence (90%) at 2-cm burial depth and emergence decreased considerably as seeding depth increased above 2 cm. Its tolerance to drought and salinity make C. biflorus a potential candidate to be used as an alternative source during periods of forage scarcity under harsh climatic conditions, and it could possibly be used for rangeland rehabilitation purposes in arid environments.     

Assessment of diclofenac LC50 reference values in juvenile and embryonic stages of the zebrafish (Danio rerio)

The aim of the study was to compare the acute toxicity of diclofenac to juvenile and embryonic stages of the zebrafish (Danio rerio). Acute toxicity tests were performed on the aquarium fish Danio rerio, which is one of the model organisms most commonly used in toxicity testing. The tests were performed using a semi-static method according to OECD guideline No. 203 (Fish, acute toxicity test). Embryo toxicity tests were performed in zebrafish embryos (Danio rerio) in compliance with OECD No. 212 methodology (Fish, short-term toxicity test on embryo and sac-fry stages). The results were subjected to a probit analysis using the EKO-TOX 5.2 programme to determine 96hLC50 and 144hLC50 (median lethal concentration, 50% mortality after a 96 h or 144 h interval, respectively) values of diclofenac. The statistical significance of the difference between LC50 values in juvenile and embryonic stages of Danio rerio was tested using the Mann-Whitney non-parametric test implemented in the Unistat 5.1 programme. The LC50 mean value of diclofenac was 166.6 +/- 9.8 mg/L in juvenile Danio rerio, and 6.11 +/- 2.48 mg/L in embryonic stages of Danio rerio. The study demonstrated a statistically higher sensitivity to diclofenac (P < 0.05) in embryonic stages compared to the juvenile fish.     

Ion,Protein, Phospholipid and Energy Substrate Content of Oviduct Fluid During the Oestrous Cycle of Buffalo (Bubalus bubalis)

The aim of this research was to analyse the composition of oviduct fluid (ODF) in buffalo cows at different oestrous cycle phases to fulfil the requirements of buffalo embryos in vitro. ODF was collected by chronic cannulation from three cows that were synchronized by administering a synthetic prostaglandin. Based on hormonal profiles, the pre‐ovulatory, ovulatory, post‐ovulatory and luteal phases of the oestrous cycle were defined. The volume of ODF produced (ml/24 h) was influenced by the oestrous cycle, with values (mean ± SE) around ovulation (1.0 ± 0.2) greater (p < 0.05) than in both the luteal (0.4 ± 0.1) and the post‐ovulatory phases (0.5 ± 0.1), but not different from the intermediate values in the pre‐ovulatory phase (0.8 ± 0.2). Among cycle phases, no differences were found in sodium, potassium, calcium and magnesium concentrations (130.0 ± 1.1, 5.1 ± 0.3, 2.8 ± 0.1 and 0.59 ± 0.04 mmol/l respectively). Interestingly, the chloride secretion (μm /24 h) was higher (p < 0.05) at ovulation (150.2 ± 16.5) than during both the luteal (73.7 ± 22.0) and the post‐ovulatory phases (63.7 ± 11.2), with intermediate values in the pre‐ovulatory phase (113.4 ± 23.5). Glucose concentration (mmol/l) was higher (p = 0.056) in the pre‐ovulatory phase (0.06 ± 0.02) than in the luteal (0.02 ± 0.01) and post‐ovulatory (0.02 ± 0.01) phases but not different from values in the ovulatory phase (0.04 ± 0.02). Concentrations of pyruvate and lactate among oestrous cycle phases were similar (0.08 ± 0.01 and 1.0 ± 0.1 mmol/l respectively). The total quantity of phospholipids (μmol/24 h) was greater (p < 0.05) at ovulation (0.21 ± 0.02) compared with the luteal, pre‐ovulatory and post‐ovulatory phases of the cycle (0.09 ± 0.02, 0.13 ± 0.02 and 0.09 ± 0.01 respectively). No differences were found in either the protein concentration (1.8 ± 0.3 mg/ml) or the quantity of proteins secreted in 24 h (1.8 ± 0.4 mg) among oestrous cycle phases. In conclusion, this study provides the first characterization of buffalo ODF during the oestrous cycle, showing species‐specific differences that may be useful for developing suitable media for buffalo in vitro embryo production.