Influences of incorporating detoxified Jatropha curcas kernel meal in common carp (Cyprinus carpio L.) diet on the expression of growth hormone‐ and insulin‐like growth factor‐1‐encoding genes

Jatropha curcas is a drought‐resistant shrub or small tree widespread all over the tropics and subtropics. The use of J. curcas (L) kernel meal in fish feed is limited owing to the presence of toxic and antinutritional constituents. In this study, it was detoxified using heat treatment and organic solvent extraction method. The detoxification process was carried out for 60 min to obtain the detoxified meal. Cyprinus carpio L. fingerlings (n = 180; avg. wt. 3.2 ± 0.07 g) were randomly distributed in five treatment groups with four replicates and fed isonitrogenous diets (crude protein 38%) for 8 weeks. The inclusion levels of the detoxified Jatropha kernel meal (DJKM) and soybean meal (SBM) were as follows: control diet was prepared with fish meal (FM) and wheat meal, without any DJKM and SBM; diets S₅₀ and J₅₀: 50% of FM protein replaced by SBM and DJKM respectively; diets S₇₅ and J₇₅: 75% of FM protein replaced by SBM and DJKM respectively. Highest body mass gain and insulin‐like growth factor‐1 (IGF‐1) gene expression in brain, liver and muscle were observed for the control group, which were statistically similar to those for J₅₀ group and significantly (p < 0.05) higher than for all other groups, whereas growth hormone gene expression in brain, liver and muscle exhibited opposite trend. Insulin‐like growth factor‐1 concentration in plasma did not differ significantly among the five groups. Conclusively, growth performance was in parallel with IGF‐1 gene expression and exhibited negative trend with GH gene expression.     

Pre-treatment of dietary plant feedstuffs with phytase and its effect on growth and mineral concentration in common carp (Cyprinus carpio L.)

Effect of phytase pre-treatment of dietary plant feed stuffs (PF) on growth and mineral concentration in common carp (C. carpio L.) was investigated. Diets used were: C0, diet with no supplement; CI, diet with incubated PF; CP0, diet supplemented with 3 g P/kg; CPI, diet with incubated PF supplemented with 3 g P/kg; Phyt0, diet fortified with 4000 U phytase/kg; PhytI, diet with PF incubated with 4000 U phytase/kg. Mean weight gain, SGR and FCR were the same (p < 0.05) in fish fed CP0, CPI or PhytI but less (p < 0.05) in other fish. Bone P was similar in fish fed CP0 (74.9), CPI (75.9) or PhytI (71.5 mg/g DM) but higher (p < 0.05) than in fish fed C0. Bone Ca and Mg were similar in fish fed CP0, CPI or PhytI but less (p < 0.05) in other fish. Bone Zn in fish fed C0, Phyt0 or PhytI was higher (p < 0.05) than that in fish fed CP0 or CPI. In conclusion, CP0, CPI and PhytI had the same effect, suggesting phytase pre-treatment as the most effective method. CPI did not have any advantage over CP0. Phytase increased mineral availability and utilization, which would minimize cost of mineral supplementation and discharges into the environment.     

Mechanism of blood coagulation in common carp (Cyprinus carpio)

In vitro, carp blood was anticoagulated by using MgSO₄ at a final concentration of 22.2 mmol L^?1 and sodium citrate at a final concentration of 11.8 mmol L^?1. The coagulation times for carp plasma diluted by ion‐free water (1:1), and that of carp plasma to which thrombocytes and small lymphocytes were added, were measured at 23 °C using standard methods, and then contrasted with the coagulation times for plasma obtained from chickens and rabbits. The shapes of the thrombocytes and small lymphocytes, which were either wet mounted or stained with hematoxylin and eosin, were observed under a light microscope. We found that: (i) the coagulation reaction of carp blood was significantly (P < 0.01) accelerated by the addition of ion‐free water; (ii) the three types of blood cells (thrombocytes, small lymphocytes and red blood cells) promoted plasma coagulation to a similar extent (P > 0.05); (iii) in carp Mg²⁺ plasma and K₂C₂O₄ plasma, the thrombocytes were usually morphologically normal, but many small lymphocytes were destroyed and became aggregated; (iv) in the citrate plasma, thrombocytes were often aggregated, but the small lymphocytes were usually morphologically normal; and (v) the coagulation time for chicken and rabbit plasma was significantly extended by adding ion‐free water.     

豌豆分离蛋白和豌豆浓缩蛋白替代部分鱼粉对鲤鱼生长性能的影响

分别采用豌豆分离蛋白和豌豆浓缩蛋白替代饲料中33%、66%和100%鱼粉(对照组鱼粉10%)配制与对照组等氮、等能、等赖氨酸、等蛋氨酸和有效磷的试验饲料(其中豌豆分离蛋白用量3%、6%和9%,豌豆浓缩蛋白用量4.5%、9%和13.5%),饲养初始体重(18.53±0.35)g的建鲤75 d。结果表明:豌豆分离蛋白替代100%鱼粉组增重和特定生长率显著高于、饲料系数显著低于对照组(P<0.05),其余处理组与对照组间差异不显著(P>0.05)。两种豌豆蛋白替代鱼粉对成活率、肥满度和肠长指数无显著影响(P>0.05),但显著影响肝胰脏指数,其中豌豆分离蛋白替代100%鱼粉组和豌豆浓缩蛋白组显著低于对照组(P<0.05)。豌豆浓缩蛋白替代100%鱼粉组可观察到对适口性有不良影响。由上述结果可得出,豌豆分离蛋白和豌豆浓缩蛋白均可作为鲤鱼料的蛋白源,本试验条件下,以生长性能为标识结合适口性推荐豌豆分离蛋白可完全替代鱼粉,适宜用量9%;豌豆浓缩蛋白可替代33%鱼粉,适宜用量4.5%。     

Optimization of Oil Extraction Process from Jatropha curcas Seed and the Analysis of Crude Protein Content in Meal

The test was aimed to optimize the oil extraction process from Jatropha curcas seed and analyzed the crude protein content in meal.Jatropha curcas seeds were selected as the experimental material,and the effects of solid to liquid ratio,temperature and extraction time on the oil extraction rate and crude protein content of meal were investigated and optimized. The results of single factor test showed that when the solid to liquid ratio was 1:18,the temperature was 40℃ and extraction time was 30 min,the oil extraction rate was highest which was the 38.22%,32.21% and 34.13%,respectively. The variance analysis results showed that the order of factors that affected the oil extraction rate were temperature > solid to liquid ratio > extraction time. The effects of temperature and solid to liquid ratio were extremely significant (P < 0.01).The extraction time did not have a significant effect on oil extraction rate (P > 0.05).The order of factors that affected the crude protein content in seed meal were temperature > extraction time > solid to liquid ratio. The effect of temperature was extremely significant (P < 0.01) and that of the extraction time was significant (P < 0.05). The results of orthogonal experiment showed that the optimal extraction conditions was A₃B₃C₃ of which the solid to liquid ratio was 1:22,temperature was 50℃ and extraction time was 50 min. The oil extraction rate was 39.41%,and the crude protein was 24.13% under this condition. This study provided the theoretical basis for the development and application of Jatropha curcas seed.     

麻疯树籽提油工艺优化及籽粕中粗蛋白质含量分析

试验旨在优化麻疯树籽提油工艺并分析其籽粕中粗蛋白质含量,以提油率、提油后籽粕中粗蛋白质含量为优化指标,探讨料液比、温度、时间3个因素对麻疯树籽提油率及籽粕中粗蛋白质含量的影响,并结合正交试验获得麻疯树籽提油的最佳工艺参数。单因素试验结果表明,料液比为1∶18、温度为40℃、提取时间30 min时,提油率达到最高,分别为38.22%、32.21%及34.13%。方差分析结果表明,各因素影响提油率的主次顺序为温度 > 料液比 > 提取时间,其中温度和料液比对提油率的影响极显著（P < 0.01）,提取时间对提油率无显著影响（P > 0.05）;影响籽粕粗蛋白质的主次顺序为温度 > 提取时间 > 料液比,其中温度对籽粕粗蛋白质含量的影响极显著（P < 0.01）,提取时间对籽粕粗蛋白质含量的影响显著（P < 0.05）。正交试验结果表明,麻疯树籽最佳提油工艺组合为A₃B₃C₃,即在料液比为1∶22、温度为50℃、时间为50 min的最佳工艺条件下,提油率为39.41%,籽粕粗蛋白质为24.13%。本研究为麻疯树籽提油及提油后籽粕的开发应用提供了理论依据。     

Prevalence and characteristics of Cyprinid herpesvirus 3 (CyHV-3) infection in common carp (Cyprinus carpio L.) inhabiting three rivers in Kochi Prefecture,Japan

Cyprinid herpesvirus 3 (CyHV-3) causes lethal disease in common and koi carp. Mortality by CyHV-3 disease has not been reported since 2011 in Kochi Prefecture, Japan. Here, we detected and quantified CyHV-3 in common carp inhabiting three rivers in the prefecture to examine if the carp are carriers of CyHV-3 as a source of infection. CyHV-3 DNA was detected in 16.7% (12/72) of brain samples in Kagami River, 3.9% (3/76) of brain and 3.9% (3/76) of gill samples in Monobe River, and 5.1% (4/79) of brain and 1.3% (1/79) of gill samples in Wajiki River. CyHV-3 genotypes identified in the 23 samples were classified as the J genotype A1 that has been found in Japan. The CyHV-3 DNA load did not differ statistically between sampling months, indicating that CyHV-3 has been silent in common carp, unlike Lake Biwa where the annual reactivation occurs in spring. Taken together, our results represented definitive evidence that seasonal changes in water temperature do not affect CyHV-3 activity in carp. Considering that infectious virus was not isolated from CyHV-3 DNA-positive samples, it was suggested that CyHV-3 establishes a latent infection in carp populations inhabiting Kagami River, Monobe River and Wajiki River. Further, the presence of circular or concatameric CyHV-3 DNA was detected in five of 23 CyHV-3 DNA-positive samples. Common carp inhabiting Lake Biwa were reported previously to harbor linear but not circular CyHV-3 DNA. This difference suggested that the CyHV-3 genome may be circularized for long-term maintenance without active viral replication.     

鲤鱼白细胞介素10基因组DNA克隆及序列分析

为获得鲤鱼白细胞介素10(interleukin 10,IL-10)全长基因组序列,本研究利用鲤鱼IL-10全长cDNA序列(GenBank登录号:JX524550),通过在两端非编码区设计引物,以提取的鲤鱼脾脏基因组为模板,使用PCR方法成功获得鲤鱼IL-10全长基因组序列。鲤鱼IL-10基因组全长2176 bp,GenBank登录号为JX524551。将所获得的序列与其他物种IL-10基因组序列相比,结果发现都含有5个外显子,4个内含子,外显子在进化上相对保守,剪切位点都符合"gt......ag"规则;序列包含540 bp的开放阅读框,编码179个氨基酸,有2段典型的IL-10氨基酸信号基序。     

Starvation-induced pathobiology in the gut of carp (Cyprinus carpio L.)

In order to assess the effects of starvation on intestinal intraepithelial lymphocytes (i-IEL) count and histological changes of the carp gut mucosa, one group of fish (n = 10) were fed commercially prepared standard diets and another group of fish (n = 10) were starved for 4 weeks. Carp starved for 4 weeks developed enteropathy, comprising folds atrophy, stratum compactum hyperplasia, significant periodic-acid-Schiff (PAS)-positive (P < 0.00001), but not Alcian blue (ALB)-positive, goblet cell (GC) hyperplasia and a significant decrease (P < 0.00001) in i-IEL numbers. These changes were associated with a dense cellular infiltrate into the lamina propria. Taken together, these data suggest that the pathobiology of starvation-induced i-IELs decrease, matching PAS-positive goblet cell proliferation and inflammatory cells homing to the gut, could be classified as a non-infectious enteropathy induced by starvation.     

Morphology of blood cells in carp (Cyprinus carpio L.)

The morphology of blood cells in the carp was investigated by light and electron microscopy. Erythrocytes, thrombocytes, lymphocytes, granulocytes and monocytes were identified as the peripheral blood cells. Thrombocytes were round to long oval, each containing vesicular and microtubular structures and an oval nucleus with abundant heterochromatins. Lymphocytes were divided into three types in size, small, medium and large. Some of the small and medium lymphocytes were alpha-naphthyl-acetate esterase (ANAE) positive, while large lymphocytes were pyroninophilic. Granulocytes were distinguished into three types (type I, type II and type III) according to the morphology of the nucleus and granules. Type I granulocytes possessed lobulated nuclei and a large number of cytoplasmic granules, each of which was oval and contained electron-dense materials and a crystalloid. Type II granulocytes had small eccentric nuclei and were subdivided into IIa and IIb granulocytes by electron microscopic analysis. Granules of type IIa granulocytes were furnished with an electron-dense rim. Granules of type IIb granulocytes were larger than those of type IIa, containing randomly distributed electron-dense and electron-lucent materials. Type III granulocytes possessed round nuclei and a few large granules. The granules were filled with regularly arranged fibriform materials and some needle-like structures. Monocytes were morphologically similar to those of mammals.     

Immunomodulatory effects of alpha melanocyte stimulating hormone on common carp (Cyprinus carpio L.)

The immunomodulatory effects of phagocytic cells in common carp Cyprinus carpio L., by alpha melanocyte stimulating hormone (alpha-MSH) was analyzed in vitro. Carp head kidney leucocytes were cultured in RPMI 1640 medium containing 0.05, 0.1, 1 and 10 nM alpha-MSH and the production of superoxide anion was measured via the reduction of nitroblue tetrazolium (NBT) in vitro. Macrophages incubated with alpha-MSH showed an increase in the production of superoxide anion in comparison to control macrophages cultured without hormone. Phagocytic cells treated with alpha-MSH also displayed increased phagocytosis. Furthermore, carp lymphocytes treated with alpha-MSH increased the mitogenic responses to phytohaemoagglutinin (PHA-P). These results show that alpha-MSH directly influences fish immune responses.     

The comparison of two different extenders for the improvement of large‐scale sperm cryopreservation in common carp (Cyprinus carpio)

In our study, a traditionally used (Grayling, already used in cyprinid species) and a newly tested (Pike) extender was tested to avoid sperm agglutination phenomenon following thawing during carp sperm cryopreservation. A large‐scale (elevated volume of sperm) freezing method in a controlled‐rate freezer using 5 ml straw and 10 ml cryotube was also systematically established. In all experiments, the sperm cryopreserved in using Grayling extender (except only one sample) showed an agglutination phenomenon (damaged and intact cells adhered to each other) after thawing where Pike extender resulted the regular cell suspension. No significant difference was observed between the two cryopreserved groups (Pike and Grayling extender) in all motility parameters using the 0.5 ml straw and the polystyrene box. Similarly, motility parameters did not show a significant difference in the two frozen groups with the 5 ml straw, also in the polystyrene box. A significantly higher progressive motility (pMOT, Grayling: 54% ± 8%, Pike: 37% ± 5%), straight line velocity (VSL, Grayling: 50 ± 5 µm/s, Pike: 39 ± 4 µm/s) and beat cross frequency (BCF, Grayling: 20 ± 1 Hz, Pike: 17 ± 1 Hz) was observed in the case of the grayling extender by the 5 ml straw cryopreserved in a controlled‐rate freezer (CRF) compare to the pike extender. A significantly higher VSL (Grayling: 45 ± 3 µm/s, Pike: 38 ± 4 µm/s) was observed by the grayling extender using the 10 ml cryotube than with the pike extender. Despite the randomly occurring differences in a few parameters, our new controlled freezing method using the newly tested Pike extender, the 5 ml straw or the 10 ml cryotube can be a good solution for the preservation of elevated volume of carp sperm.     

不同处理对麻疯树籽仁粕蛋白质营养价值的影响

试验旨在研究不同处理对麻疯树籽仁粕（Jatropha curcas kernel meal,JCKM）蛋白质营养价值的影响。选择脱油后的JCKM为研究对象,对其分别进行干热、湿热、热化学和微生物发酵处理,通过测定其营养成分、蛋白质溶解度（PS）、粗蛋白质体外消化率（IVCPD）等指标,以未处理的JCKM为对照,评价不同处理对JCKM蛋白质营养价值的影响。结果显示：微生物发酵处理组粗蛋白质（CP）含量显著高于其他组（P < 0.05）;微生物发酵处理组粗纤维（CF）含量显著低于热化学处理组（P < 0.05）,其他各组间差异不显著（P > 0.05）;各处理组间钙（Ca）含量差异不显著（P > 0.05）;微生物发酵处理组总磷（TP）含量最高,显著高于其他组（P < 0.05）。微生物发酵处理组Gly、Arg、Cys、Val、Ile含量高于其他组。干热处理、微生物发酵处理及对照组PS显著高于湿热处理和热化学处理组（P < 0.05）;微生物发酵处理组IVCPD显著高于其他组（P < 0.05）。综上所述,微生物处理JCKM后CP含量升高,氨基酸组成较为均衡,是一种有效的处理方法。     

螺旋藻对锦鲤生长和养分消化率的影响

在基础饲料中分别添加5种不同含量的螺旋藻(0%、4%、8%、12%、16%)饲养锦鲤60 d,研究其对锦鲤生长和养分消化率的影响。实验结果显示,随着螺旋藻添加量的不断增加,实验组锦鲤的增重率、肝体比指数有显著增加(P<0.05),但对特定生长率、肥满度、内脏比等影响不显著(P>0.05)。随着螺旋藻添加含量的增加,实验组的干物质消化率、蛋白质消化率、脂肪消化率也逐渐升高,均显著高于对照组(P<0.05)。     

Jatropha platyphylla kernel meal as feed ingredient for Nile tilapia (Oreochromis niloticus L.): growth, nutrient utilization and blood parameters

Jatropha platyphylla is a multipurpose and drought-resistant shrub, available in Mexico, locally known as 'sangregrado' and belonging to the family Euphorbiaceae. Its seeds are rich in oil and protein and do not contain the main toxin, phorbol esters present in other Jatropha species. Jatropha platyphylla kernel meal (JPKM) obtained after oil extraction contained 70-75% crude protein (CP); however, it contained phytate, lectin and trypsin-inhibitor. The levels of essential amino acids (except lysine) were higher in JPKM than in soybean meal (SBM). Using Nile tilapia (Oreochromis niloticus) fingerlings a 12-week experiment was conducted to evaluate the nutritional quality of the heated JPKM and compare with that of SBM and fishmeal. Fingerlings (15 fish; average weight 13.7 ± 0.21g) were randomly distributed in three treatment groups with five replicates. Fish were fed three isonitrogenous diets (CP 36%): control diet containing fishmeal-based protein and two other diets replacing 62.5% fishmeal protein with JPKM (Jatropha group) and SBM (Soybean group). The growth performance, feed conversion ratio, protein efficiency ratio, protein productive and energy retention did not differ significantly among the three groups. A lower apparent lipid conversion was observed in the plant protein-fed group than in the control group. RBC count, haematocrit and blood glucose contents were higher in plant-protein fed groups than control group. Other haematological parameters (WBC count, haemoglobin, mean cell volume: calcium and sodium ions, total bilirubin and urea-nitrogen in the blood) and metabolic enzymes (alkaline phosphatase and alanine transaminase) activities in blood did not differ significantly among the three groups. The results from the present study established that JPKM is a promising and good quality protein source for Nile tilapia feed.     

Immunosuppression of carp (Cyprinus carpio L.) to ichthyophthiriasis using the corticosteroid triamcinolone acetonide

The immune response of juvenile mirror carp (Cyprinus carpio L.) has been investigated in relation to protective immunity and immunosuppression to the disease ichthyophthiriasis. Protective immunity was induced by exposing juvenile carp to approximately 2,000 theronts per fish for 3 hours at a concentration of approximately 80 theronts cm-3 in dechlorinated water pH 7.0-7.2, 20 +/- 2 degrees C in the dark, on three separate occasions of 14 day intervals. Following each exposure, re-infection was prevented by transferring fish to clean aquaria twice daily from day 5-10. Fish were challenged 4 weeks following exposure to the third immunising infection with a potentially lethal dose of approximately 8,000 theronts per fish for 3 hours at a concentration of approximately 320 theronts cm-3. All immunised fish survived although 19% showed initial signs of invasion, the parasites in these instances being confined to periphery of fins. Mortalities of 100% were recorded in non-immunised controls, first deaths occurring 5 days after challenge. Fourteen days following challenge, immune fish received one of the following treatments; intraperitoneal injection of the corticosteroid triamcinolone acetonide at a dose of 200 micrograms g-1 body weight, intraperitoneal injection of 0.85% saline or left untreated; non-immunised fish were subjected to similar procedures. On challenge 6 days later, all fish developed heavy infection with up to 100% mortalities with the exception of immunised fish administered saline or left untreated. Repeat experiments gave comparable results. The results indicate that immunosuppression can be induced by administration of high levels of synthetic corticosteroid.(ABSTRACT TRUNCATED AT 250 WORDS)     

锦鲤溃疡病病原菌的分离及16SrRNA鉴定

为寻找引起养殖锦鲤(Ornamental carp)病害的致病因子,从北京地区自然患病的锦鲤体内分离疑似致病菌,再将此菌人工感染健康锦鲤,确定致病菌。采用生理生化鉴定与16S rRNA基因序列的系统发育学分析相结合的方法确定该致病菌株的系统发育地位,同时采用琼脂扩散法测定该菌对抗菌类药物的敏感性。试验结果表明,从病鱼体内分离得到革兰氏阴性杆菌CL0901,人工感染健康锦鲤后,能够引起鱼生病甚至死亡,症状与自然发病症状一致。菌株CL0901与Aeromonas veronii ATCC 35624T的16S rRNA基因序列相似性达99.9%,构建系统发育树,并结合形态特征与生理生化测定结果,将该致病菌鉴定为维氏气单胞菌(Aeromonas veronii)。在21种抗菌类药物对该菌的抑菌试验中,左氧氟沙星、诺氟沙星和洛美沙星等10种药物的抑菌效果较好。本研究为进一步防制锦鲤养殖病害提供依据。     

Ovarian development and related changes in steroid hormones in female wild common carp (Cyprinus carpio carpio), from the south‐eastern Caspian Sea

Wild common carp (Cyprinus carpio carpio) is a native valuable but threatened species from the south‐eastern Caspian Sea in which the endocrine control of its reproduction has not been studied. The objectives of this research were to study the reproductive strategy and changes in steroid hormones during ovarian development. From October 2009 to June 2010, 65 adult females were caught from the Golestan coast (Iran) and the ovarian histology, and gonadosomatic and hepatosomatic indices (GSI and HSI) were studied. Also, the plasma profiles of steroid hormones including testosterone (T), 17β‐estradiol (E2) and 17α‐, 20β‐dihydroxyprogesterone (DHP) were measured by radioimmunoassay. The GSI increased gradually during sampling times and reached maximum value at the peak of reproduction season, but the HSI decreased during spawning season. All stages of ovarian development, except the stage of Balbiani bodies, were recorded macro‐ and microscopically. Spent fish were caught at six of nine sampling times. The peaks of spawning were at late winter and early spring. The results of this study showed that the majority of wild carp collected during the sampling period displayed asynchronous oocyte development. Plasma T showed no significant differences during sampling times or at different stages of ovarian development. The level of E2 decreased gradually during sampling times reached minimum value at the spawning season, and highest value was recorded at tertiary vitellogenesis stage. The plasma levels of DHP during late winter and early spring were significantly higher than those of other sampling periods and its maximum level associated with oocyte maturation stage.