硫化物胁迫对凡纳滨对虾血细胞的毒性影响

硫化物是水产养殖过程中常见的水体污染物之一。为探讨水体硫化物胁迫对虾类血细胞的毒性影响,以不同浓度(0.5 mg/L,2.0 mg/L)的硫化物对凡纳滨对虾(Litopenaeus vannamei)进行胁迫,于胁迫后6、12、24和48 h取血淋巴,应用流式细胞术测定血细胞的总数(THC)、活性氧(ROS)含量、一氧化氮(NO)含量以及凋亡率。结果显示,经0.5 mg/L硫化物胁迫48 h后,对虾THC显著下降至11.78×10~6个/mL,为对照组的72.7%(P0.05),ROS含量为对照组的225.2%(P0.05),血细胞凋亡率显著上升至7.42%(P0.05);经2.0 mg/L硫化物胁迫6 h开始,对虾THC呈现显著的下降(P0.05),血细胞ROS含量和凋亡率显著提高(P0.05);表明硫化物胁迫刺激对虾血细胞产生大量ROS,诱导血细胞凋亡,从而导致THC下降,这一过程可能是硫化物导致虾类细胞免疫下降的重要机制;随着硫化物浓度的升高,其细胞毒性作用明显提高。血细胞NO含量在2.0 mg/L硫化物胁迫12和48 h时显著升高(P0.05),推测NO可能在硫化物胁迫防御调控中起着信号因子的作用。     

亚硝酸盐对凡纳滨对虾血细胞毒性及p53基因表达的影响

随着水产动物集约化养殖的迅猛发展,水质污染问题日益加剧,环境中亚硝酸盐的含量不断上升,成为水产养殖中诱发爆发性疾病的重要环境因子。为探讨亚硝酸盐对凡纳滨对虾（Litopenaeus vannamei）血细胞的毒性以及 p53 mRNA 的影响,通过预试验以及前期资料获取亚硝酸盐对凡纳滨对虾的毒性效应,选取0 mg／L 对照组和20 mg／L 浓度组,用流式细胞术检测血细胞活性氧（ROS）、一氧化氮（NO）含量和非特异性酯酶活性,运用荧光定量 PCR 技术检测 p53基因表达量变化。结果显示,在亚硝酸盐应激12 h 后,对虾血细胞的 NO 含量与对照组相比有显著升高（P ＜0．05）,在应激24、48、72 h 后有极显著的升高（P ＜0．01）;ROS 含量在应激12、48、72 h 时有极显著升高（P ＜0．01）,在应激24 h 时与对照组相比有显著的升高（P ＜0．05）。非特异性酯酶活性在应激24、48、72 h 显著下降（P ＜0．05）,p53的表达水平在应激48 h 和72 h 后显著升高（P ＜0．05）。研究表明,亚硝酸盐应激诱导对虾血细胞产生了过量的 NO 和 ROS,造成氧化胁迫作用,诱导凋亡相关基因的表达,最终导致细胞凋亡。     

低鱼粉饲料中添加羟基蛋氨酸硒对凡纳滨对虾生长性能、抗氧化能力和抗亚硝酸盐胁迫的影响

[目的]本实验旨在研究低鱼粉饲料 中添加羟基蛋氨酸硒(HMSe)对凡纳滨对虾(Litopenaeus vannamei)生长性能、抗氧化能力及抗亚硝酸盐胁迫的影响。[方法]实验选用初始体质量为(0.90±0.05)g的凡纳滨对虾,分别投喂HMSe添加水平为0.000、0.375、0.750、1.500和2.250 mg/kg的实验饲料,命名为HMSe0 、HMSe1、HMSe2、HMSe3和HMSe4,每组3个重复,养殖8周。养殖实验结束后,进行12 h 的亚硝酸盐胁迫实验。[结果]结果表明,凡纳滨对虾的终末体质量(FBW)、增重率(WG R)和饲料效率(FE)随着饲料中HMSe水平的增加呈现先升高后下降的趋势,并在HMSe2组达到峰值。随着饲料中HMSe水平的提高,对虾全体和肌肉中的硒含量均显著增加,而全虾粗蛋白和粗脂肪的含量呈现先升高后降低的趋势,并分别在HMSe2组和HMSe1组达到最大值。HMSe4组中凡纳滨对虾的血清谷胱甘肽过氧化物酶(GSH-Px)和肝胰腺过氧化氢酶(CAT)活性显著高于HMSe0组。亚硝酸盐胁迫显著影响凡纳滨对虾的存活率。与胁迫前比较,胁迫后对虾肝胰腺中MDA含量升高,GST、CAT和T-SOD的活性降低,而补充适量的HMSe可减轻这些负面影响。根据凡纳滨对虾胁迫后成活率的二次多项式回归分析,低鱼粉饲料中凡纳滨对虾的HMSe最适添加量为1.350 mg/kg。[结论]综上所述,在低鱼粉饲料中添加0.750~1.350 mg/kg HMSe,能够提高凡纳滨对虾的生长性能、饲料利用率和抗亚硝酸盐胁迫的能力。     

饲料中添加桑叶黄酮对凡纳滨对虾生长性能、抗氧化指标及抗胁迫能力的影响

为了探讨桑叶黄酮对凡纳滨对虾（Litopenaeus vannamei）生长性能、体成分、血清生化和抗氧化指标的影响，选用初始体重为（1.32±0.01）g的凡纳滨对虾960尾，随机分为6组（每组4个重复，每个重复40尾），分别投喂在基础饲料中添加0、10 mg/kg、50 mg/kg、100 mg/kg、150 mg/kg和300 mg/kg桑叶黄酮的实验饲料，饲养50 d后测定成活率、生长相关指标、血清生化指标、抗氧化指标及抗低氧胁迫能力。结果显示，饲料中添加桑叶黄酮对凡纳滨对虾成活率、增重率、特定生长率、饲料系数等无显著影响（P>0.05）。饲料中添加桑叶黄酮对凡纳滨对虾体成分无显著性影响（P>0.05）。添加150 mg/kg和300 mg/kg桑叶黄酮可显著提高凡纳滨对虾血清谷丙转氨酶和谷草转氨酶活性（P<0.05）。添加10~300 mg/kg桑叶黄酮可显著提高血清和肝脏总抗氧化能力，显著降低肝胰腺丙二醛和脂质过氧化物的含量，添加50 mg/kg桑叶黄酮可提高血清谷胱甘肽过氧化物酶的活性（P<0.05）。低氧胁迫2 h时，10 mg/kg、50 mg/kg组的累计死亡率显著低于对照组（P<0.05）；在低氧胁迫4 h时，10 mg/kg、50 mg/kg、100 mg/kg组的累计死亡率显著低于对照组（P<0.05）。结果表明，以血清总抗氧化能力为评价指标进行回归分析得出，桑叶黄酮在凡纳滨对虾饲料中的适宜添加量为56.18 mg/kg，添加10~100 mg/kg桑叶黄酮可提高凡纳滨对虾抗低氧胁迫能力。     

饲料铜的添加量对南美白对虾生长、血液免疫因子及组织铜的影响

通过在饲料中分别加入不同水平的硫酸铜,初步研究了饲料铜的添加量与南美白对虾(Litopenaeusvannamei)生长、血液免疫因子及组织中铜分布的关系。饲料铜元素的添加量分别为0、7.5、15、30、45mg/kg。研究结果表明,铜的含量对南美白对虾生长影响显著,添加水平30mg/kg的实验组生长速度显著高于其他组(P<0.05),成活率各组间差异不显著。实验组对虾的酚氧化酶(PO)和超氧化物歧化酶(SOD)的活力都高于对照组,其中当添加水平为30mg/kg时,两种酶的活力均最强。对虾肌肉铜的含量各组间无显著差异,为(4.27±1.83)mg/kg,个体间差异非常大;肝胰脏(包括淋巴器官)铜的最高含量为210.36mg/kg,最低为33.78mg/kg(空白对照组),明显高于肌肉中的含量。研究结果表明,饲料铜的含量对对虾生长影响显著,并影响对虾的免疫能力;肝胰脏是对虾体内铜的主要积累器官。     

家蝇抗菌肽对凡纳滨对虾生长性能及免疫相关指标的影响

通过8周的生长实验研究了饲料中添加家蝇幼虫抗菌肽提取物对初始体质量为(0.86±0.01)g的凡纳滨对虾(Litopenaeus vannamei)的生长性能、体成分及免疫相关指标的影响。家蝇抗菌肽提取物添加量分别为0、1000mg/kg、2000mg/kg、3000mg/kg、4000mg/kg、5000mg/kg,每组饲料设4个重复,每个重复饲养40尾虾。结果显示,在一定添加水平范围内,家蝇抗菌肽能显著提高凡纳滨对虾成活率、增重率、特定生长率、饲料效率(P0.05),当抗菌肽提取物添加量为2000～3000mg/kg时,以上4个指标均达最高,且显著高于对照组(P0.05)。以增重率为评价指标,凡纳滨对虾饲料中家蝇抗菌肽提取物的最适添加量为2900mg/kg。当抗菌肽提取物添加量为2000～3000mg/kg时能显著提高虾体的粗蛋白和灰分含量,虾体粗脂肪不受添加抗菌肽水平的显著影响。添加2000～5000mg/kg抗菌肽提取物组与对照组相比能显著提高对虾血细胞数量;添加3000mg/kg抗菌肽提取物时对虾血细胞吞噬率最高。当抗菌肽提取物添加量为2000～3000mg/kg时,对虾血清中酚氧化酶PO、过氧化物酶POD、碱性磷酸酶AKP、溶菌酶LZM的活性和总抗氧化能力T-AOC均维持在一个较高的水平,且显著高于对照组;超氧化物歧化酶SOD的活性在各组组间差异不显著(P0.05)。肝胰腺中AKP、SOD、T-AOC活性各组间差异均不显著(P0.05);LZM活性以3000mg/kg组最高,显著高于其他组。结果表明,饲料中添加适量的家蝇抗菌肽对凡纳滨对虾有一定的促生长作用,并能提高对虾的免疫相关指标。     

高铜饲料对凡纳滨对虾生长、肌肉和肝胰脏铜积累的影响

采用在实用饲料(含铜11.68 mg/kg)中分别添加铜(以分析纯CuSO4.5H2O为铜源)了0 mg/kg(对照组),25 mg/kg(适量组),50、100、200、400 mg/kg(高铜组)(编号为C0、C25、C50、、C100、C200、C400)的6组饲料,喂养凡纳滨对虾(Litopenaeus vannamei)(0.49±0.03 g)8周,研究了高铜饲料对凡纳滨对虾生长、肌肉和肝胰脏铜积累的影响。结果显示:饲料铜含量过高具有毒性,C100~C4003组存活率显著低于其它组(P<0.05),分别为51.11%、37.78%、14.81%;随饲料铜水平的增加,增重率、饲料效率、特定增长率先升高后降低,C50组的增重率和特定生长率显著高于其他组(P<0.05),C25、C50组的饲料效率显著高于其他组(P<0.05);随饲养时间的延长,肌肉、肝胰脏铜积累增加,试验末期各组肌肉、肝胰脏铜积累显著升高(P<0.05),试验期间肌肉铜积累在3.10~11.08 mg/kg之间,而肝胰脏铜积累则在10.89~449.48 mg/kg之间,同组对虾肝胰脏铜积累量远高于肌肉;饲养8周肌肉铜积累小于50 mg/kg,符合食品安全,而肝胰脏除C0组外均大于50 mg/kg。     

抗菌肽对凡纳滨对虾生长和血清非特异性免疫指标的影响

为考察抗菌肽对凡纳滨对虾(Litopenaeus vannamei)生长、饲料利用和免疫能力的影响,分别在基础组饲料中添加0 mg/kg(对照组)、300 mg/kg、400 mg/kg和500 mg/kg抗菌肽,投喂平均体重为(0.8±0.1 g)的凡纳滨对虾6周。结果表明,饲料中添加抗菌肽后,凡纳滨对虾的成活率均显著高于对照组(P<0.05);300 mg/kg抗菌肽组的增重率和饲料系数分别为509.10%、1.33,较对照组提高增重率8.76%(P<0.05),降低饲料系数12.5%(P<0.05),而添加400 mg/kg、500 mg/kg抗菌肽对增重率、饲料系数无显著影响,但显著提高了肌肉粗脂肪含量,各处理组在肌肉粗蛋白、水分含量上无显著差异;300 mg/kg、400 mg/kg抗菌肽组的血清碱性磷酸酶、超氧化物歧化酶、谷丙转氨酶、谷草转氨酶及400 mg/kg抗菌肽组的溶菌酶均显著高于对照组。上述研究表明,饲料中添加300 mg/kg抗菌肽,可显著提高凡纳滨对虾成活率和增重率,降低饲料系数;添加400 mg/kg抗菌肽,对血清非特异性免疫指标有提高作用,凡纳滨对虾饲料中抗菌肽的添加量建议为300～400 mg/kg。     

应用流式细胞术检测虾类血细胞总数方法的建立

通过对流式细胞术(FCM)检测虾类血细胞总数(THC)的条件和方法进行优化,为虾类血细胞学研究提供快捷、准确的测定方法。2012年7-9月,应用SYBR Green I作为荧光染料标记完整血细胞,设置3个不同染料浓度(1×、10×和100×),测定不同孵育时间下染色细胞比例的变化。结果显示,染料终浓度为10×时染色效果最佳,其最佳孵育时间为60 min;应用建立的FCM方法和显微计数方法测定10尾凡纳滨对虾(Litopenaeus vannamei)的THC,平均值分别为(16.68±1.57)×106个/m L和(15.09±1.76)×106个/m L,2种方法测定结果的相关性极显著(R2=0.8064,P<0.01)。凡纳滨对虾经不同浓度(0.5 mg/L和5.0 mg/L)的Cd2+胁迫,利用建立的FCM方法测定Cd2+胁迫下对虾THC的变化。结果显示,0.5 mg/L和5.0 mg/L Cd2+胁迫下,对虾THC随着胁迫时间的延长不断下降,胁迫48 h时分别下降至对照组的78.7%和64.7%,可见Cd2+胁迫对虾类血细胞产生毒性,抑制了血细胞活性,表明该方法适用于虾类的血细胞学研究。     

急性氨氮胁迫对红螯螯虾血细胞凋亡的影响

氨氮是水产养殖过程中的常见毒性污染物之一,为探讨氨氮对红螯螯虾(Cherax quadricarinatus)血细胞的细胞毒性机制,研究了氨氮胁迫作用下红螯螯虾血细胞凋亡状态的变化。以17.6mg/L氨氮对红螯螯虾幼虾进行急性胁迫,于胁迫后的0、48、72和96h取血淋巴,应用流式细胞术测定血细胞总数(THC)、胞内游离Ca~(2+)含量、线粒体膜电位(MMP)和血细胞凋亡率。结果显示,红螯螯虾的THC在胁迫的72h开始有显著的下降,在96h时达到最低值;血细胞胞内游离Ca~(2+)含量在48h开始有显著的升高,在96h达到最高值;血细胞MMP在72h开始有显著的下降,在96h达到最低值;血细胞凋亡率在72h开始有显著的升高,在96h时达到最高值,为9.39%。这些结果表明,胞内游离Ca~(2+)含量是对氨氮胁迫最为敏感的指标;氨氮急性胁迫诱导红螯螯虾血细胞发生了Ca~(2+)参与调节、线粒体通路相关的细胞凋亡,从而导致了THC的下降。     

Dietary folic acid requirement determined for grass shrimp, Penaeus monodon

A feeding trial was conducted to determine the dietary folic acid requirement of juvenile grass shrimp, Penaeus monodon. Purified basal diets were formulated using vitamin-free casein as the protein source. Graded levels (0, 1, 2, 5, 10, 15, 30, and 60 mg folic acid/kg diet) of folic acid were added to the basal diet, resulting in eight dietary treatments in the experiment. Each diet was fed to three replicate groups of shrimp initially averaging 0.79±0.01 g for 8 weeks. Shrimp fed diets supplemented with 2 mg folic acid/kg diet had greater (P<0.05) weight gain and hepatopancreatic folic acid concentrations than shrimp fed the unsupplemented control diet. Feed efficiency (FE) was highest in shrimp fed the diets supplemented with 2 mg folic acid/kg diet, followed by the groups fed 1 mg/kg, then the control group. Hepatosomatic index (HSI) was highest in shrimp fed the control diet, followed by 1 mg folic acid/kg and lowest in shrimp fed diets supplemented with 2 mg folic acid/kg. Weight gain percentage, hepatopancreatic folic acid concentration and hepatosomatic index of the shrimp analyzed by broken-line regression indicated that the adequate dietary folic acid concentration in growing P. monodon is 1.9–2.1 mg/kg diet.     

Evaluation of the Dietary Zinc Requirement of Penaeus vannamei and Effects of Phytic Acid on Zinc and Phosphorus Bioavailability

Experiments were conducted to determine the dietary zinc requirement of Penaeus vannomri and evaluate the effects of phytate on zinc bioavailability. Prior to initiation of the growth trial, 20-day-old P. vonnamei postlarvae (mean weight 0.0032 g) were fed a casein-gelatin based semi-purified diet lacking zinc supplementation but containing 18 mg Zn/kg diet for one week. Subsequently, juveniles (mean weight 0.058 g) were fed one of seven diets containing either supplemental zinc (0, 15, 30, 60 mg/kg diet) without phytate or supplemental zinc (0, 60, 200 mg/kg diet) with 1.5% phytate for 33 days. Weight gain was greatest in shrimp fed 15 mg supplemental Zn/kg diet. In the absence of dietary phytate, zinc concentrations in the hepatopancreas of shrimp were maximized when zinc was supplemented at levels greater than or equal to 15 mg Zn/kg diet (33 mg total Zn/ kg). Supplementation of 1.5% phytate to the diet did not have a significant effect on growth or zinc concentrations in the carapace; however, it did depress zinc levels in the hepatopancreas. Supplementation of 200 mg Zn/kg diet was required to overcome the depressed bioavailability of zinc caused by the presence of dietary phytate and return zinc levels of the hepatopancreas to that observed when phytate was not present. Based on apparent digestibility values phytate phosphorus was unavailable to the shrimp and the presence of phytate depressed the bioavailability of phosphorus and zinc.     

A Comparison between dietary effects of Cuminum cyminum essential oil and Cuminum cyminum essential oil,loaded with iron nanoparticles,on growth performance,immunity and antioxidant indicators of white leg shrimp (Litopenaeus vannamei)

This study evaluates the effect of dietary cumin essential oil's (CEO) and cumin essential oil's loaded iron nanoparticles (CEO‐loaded NPs) on growth performance, haematological parameters and antioxidant status of white leg shrimp. Shrimps with the initial weight of 3.38 ± 0.014 g were allocated into 15 fibreglass tanks (300 L, 60 shrimps per tank) and were fed with five experimental diets containing 0 g/kg CEO (the control diet), 5 g/kg CEO, 10 g/kg CEO, 5 g/kg CEO‐NPs and 10 g/kg CEO‐NPs for 56 days. After 56 days of culture, the shrimps fed with CEO and CEO‐loaded NPs supplemented diets showed a more significant increase in growth performance (p < .05) than in the control diet. Total haemocyte count (THC) value, large‐granule cells (LGC), hyaline cells (HC)%, superoxide dismutase (SOD) and Alkaline phosphatase (AKP) activity were significantly higher (p < .05) in the shrimps fed with CEO and CEO‐loaded NPs supplemented diets than the shrimps fed with the control diet. Glutathione peroxidase (GPX) enzyme activities along with the glucose and cholesterol concentrations significantly decreased in the shrimps fed with the CEO and CEO‐loaded NPs supplemented diets more than those fed with the control diet. The results suggest that the dietary CEO‐loaded NPs can improve the growth performance, the health status and the antioxidant capacity of L. vannamei.     

饲料中不同泛酸水平对凡纳滨对虾生长性能、饲料利用及血清指标的影响

为研究饲料中不同泛酸水平对凡纳滨对虾生长性能、饲料利用及血清指标的影响,实验配制6种等氮等脂(41%粗蛋白质和8%粗脂肪)的实验饲料,泛酸添加水平分别为0(对照组)、50、100、150、300和600 mg/kg,饲料中实测泛酸水平分别为20.9、69.3、99.0、150.2、304.4和513.6 mg/kg。选用初始体质量为(0.73±0.12)g的凡纳滨对虾720尾,随机分为6组(每组3个重复,每个重复40尾),进行为期8周的养殖实验。结果显示,饲料中不同泛酸水平对凡纳滨对虾增重率和特定生长率的影响不显著;对照组凡纳滨对虾成活率显著低于泛酸添加组,然而各泛酸添加组的成活率组间差异不显著。随着泛酸水平从20.9 m.g/kg增加到99.0 mg/kg时,饲料效率和蛋白质效率显著提高,而随着泛酸水平的进一步增加,饲料效率和蛋白质效率显著降低。饲料中泛酸水平对凡纳滨对虾全虾和肌肉主要成分无显著性影响。对照组对虾血清葡萄糖含量显著低于泛酸各添加组,然而泛酸各添加组之间无显著差异;投喂150.2 mg/kg泛酸组血清甘油三酯含量显著低于对照组和513.6 mg/kg;而血清总蛋白和胆固醇含量不受饲料中泛酸水平的影响。随着饲料中泛酸水平由20.9 mg/kg增至150.2 mg/kg,血清超氧化物歧化酶活力显著提高,而随着饲料中泛酸水平的进一步增加,血清超氧化物歧化酶活力显著降低;饲料中泛酸水平为99.0mg/kg组凡纳滨对虾血清总氧化能力显著高于其他各组;投喂对照组和泛酸水平为69.3mg/kg组的凡纳滨对虾血清丙二醛含量最高,投喂泛酸水平为150.2 mg/kg组血清的丙二醛含量显著低于其他各组。以凡纳滨对虾的饲料效率和蛋白质沉积率为评价指标,通过折线模型分析得到凡纳滨对虾泛酸最适需要量分别为113.40和119.87mg/kg。     

Oral Administration of Hot‐water Extract of Tropical Brown Seaweed,Sargassum cristaefolium,to Enhance Immune Response,Stress Tolerance,and Resistance of White Shrimp,Litopenaeus vannamei,to Vibrio parahaemolyticus

The efficacy of hot‐water extract of tropical brown seaweed, Sargassum cristaefolium (SCE), supplemented in diets on immune response, stress tolerance, and disease resistance of Litopenaeus vannamei to Vibrio parahaemolyticus was evaluated. Shrimp were fed diets containing graded levels of SCE (0, 250, 500, 750, and 1000 mg/kg). The results showed that shrimp fed all diets containing SCE had significantly higher (P < 0.05) immune response in total hemocyte count (THC), differential hemocyte count (granular and hyaline cells), and phagocytic activity than those of shrimp fed the control diet. Similarly, in low dissolved oxygen stress tolerance test and the challenge test with V. parahaemolyticus, survival rates of shrimp fed all diets containing SCE were significantly higher (P < 0.05) (83–93% in stress test and 27–47% in challenge test) than those of shrimp fed the control diet (77 and 3.3%, respectively). These results suggest that oral administration of SCE at 500 and 750 mg/kg can be effectively used to enhance immune response, stress tolerance, and resistance of white shrimp, L. vannamei, against V. parahaemolyticus infection. These findings also confirm that using dietary SCE as immunostimulant is effective at increasing the nonspecific immune system in penaeid shrimp, L. vannamei.     

Physiological, metabolic and haematological responses in white shrimp Litopenaeus vannamei (Boone) juveniles fed diets supplemented with astaxanthin acclimated to low-salinity water

The effects of dietary astaxanthin supplemented at 0, 40, 80 or 150 mg astaxanthin kg⁻¹ on growth, survival, moult frequency, osmoregulatory capacity (OC) and selected metabolic and haematological variables in Litopenaeus vannamei acclimated to low‐salinity water (3 g L⁻¹) were evaluated. Supplemented astaxanthin at 80 mg kg⁻¹ improved growth, survival and moult frequency in shrimp. The lowest OC was also exhibited in shrimp fed with dietary astaxanthin at 80 mg kg⁻¹. Shrimp haemolymph concentrations of glucose, lactate, haemocyanin and total haemocyte count were all significantly enhanced by feeding the diet supplemented with 80 mg astaxanthin kg⁻¹ compared with shrimp fed with the other diets. On the basis of these results, dietary astaxanthin supplementation of 80 mg kg⁻¹ is recommended for juvenile L. vannamei cultured in low‐salinity water.