Hemolymph Clotting Time Research Articles

Clinical and histopathological evidence of Hepatobacter penaei in Litopenaus vannamei culture with high mortality

With the appearance of high mortalities in traditional cultivation of Pacific white shrimp (Litopenaus vannamei), the objective was to evaluate the health in semi-intensive nurseries in Mossoró/RN. A random sample of 200 shrimp was collected for presumptive clinical and histopathological examination of the hepatopancreas. Histologically, the hepatopancreatic fragments were evaluated for the presence of intracytoplasmic bacteria, and scores (1-4) were assigned according to the degree of severity of the lesions (DS). Morbidity percentages of 89.5% of shrimp infected with the bacterium Hepatobacter penaei were recorded. Hemolymph clotting time did not prove to be a good parameter to diagnose individuals with necrotizing hepatopancreatitis. The affected individuals presented melanized lesions; 45.5% presented focal to diffuse necrosis in the abdominal segments, and the pleopods presented an expansion of chromatophores only in the most evolved cases of the infection, with 73% presenting uropods with expanded chromatophores. In the hepatopancreas, it was found that 57.5% had tubules with a significant reduction in lipid levels, 66.5% had DS 3 indicating the transition phase of the disease, and 23% had DS 4 considering the chronic phase. Absence of cytoplasm was observed in B cells with chromatin condensation. It is concluded that abiotic factors such as salinity and high temperatures favored the virulence of the pathogen in marine shrimp ponds, triggering a high mortality rate.

Dietary lacto-sacc stimulates the immune response of gravid mud crab (Scylla olivacea)

Mud crabs have a functioning innate immune system. Probiotics have a considerable effect in improving immune responses in gravid mud crab. The current study investigated the influence of three lacto-sacc concentrations (T1=0 %, T2=1 %, and T3=1.5 %) in a 45 % protein and 12 % lipid meal on the immune response of gravid mud crabs. Three groups of wild gravid mud crabs (average weight: 122.33±1.53 g) were raised individually in bamboo spawning boxes at mangrove pens for 12 weeks. Wild gravid mud crabs (T4) were also caught from the river during the breeding season and tested to offer a more exact assessment of the effect of lacto-sacc. After a 12-week feeding study, T3 therapy resulted in the highest total hemocyte count levels (P < 0.05), followed by T2 and T1. The T3 treatment results in somewhat faster hemolymph clotting, although there is no significant difference among the treatments (P < 0.05). T3 treatments significantly improved immunological parameters (PO, proPO, and SOD enzyme) and survival (61.67±2.89 %) when challenged with Vibrio parahaemolyticus. In the challenge test, the immunological markers THC, PO, and proPO correlated positively, but hemolymph clotting time and mortality linked negatively. Furthermore, T1 was compared to an untreated wild gravid female mud crab (T4), and the results were almost identical. Thus, 1.5 % lacto-sacc is recommended for gravid mud crabs.

SESBANIA GRANDIFLORA LEAF EXTRACT ENHANCES GROWTH AND NON-SPECIFIC IMMUNE RESPONSES OF SHRIMP (Penaeus monodon)

This study determined the effects of Sesbania grandiflora (SG) leaf extract on the growth, feed utilization efficiency and non-specific immunological responses in black tiger shrimp (Penaeus monodon). Three diets were prepared by mixing the methanol extract of SG onto a pellet feed (34% protein) at concentrations of 0% (control), 0.05% (T1) and 0.1% (T2), respectively. Ten shrimps (mean weight 2.1g), were reared in each tank of 80 L, and the weight and immunological reaction of shrimp were received after a feeding trail of 28 days. The dietary extract treated shrimp had better growth performance (WG, SGR), and feed utilization efficiency (FCR, PER) compared to the control group. The treated shrimp exhibited enhanced immunological responses; the total haemocyte count, prophenoloxidase activity, superoxide dismutase activity, and shorter haemolymph clotting time compared to the control shrimp. In conclusion, a lower level of SG (0.05%) was found to be most effective in terms of immune response enhancement; hence, this plant could be a good source for feed additives for enhancing sustainable shrimp production.

THE IMPACT OF VASAKA (JUSTICIA ADHATODA) LEAF EXTRACT ON THE GROWTH AND IMMUNE RESPONSE OF SHRIMP (PENAEUS MONODON)

Justicia adhatoda, also known as "Vasaka plant," is employed in Ayurvedic medicine to treat a wide range of human diseases. This study aimed to explore the potential application of Vasaka leaf extract (VLE) as a viable option for enhancing the growth and immune response of Penaeus monodon. Three diets containing varying amounts of Vasaka leaves (0 g [Control], 0.5 g/kg feed [VLE0.5], and 1 g/kg feed [VLE1.0]) were consumed to replicate groups of shrimp in 60 L aquarium tanks for 28 days. The assessment of growth was conducted by the evaluation of many parameters, including weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio and survival rate. The results of the experiment indicate that there were no statistically significant differences in the growth performance and survival rate of the shrimp across the various experimental groups. The introduction of Vasaka leaf as a dietary supplement resulted in a significant enhancement of shrimp immunity through the upregulation of total haemocyte count, prophenoloxidase activity, and superoxide dismutase activity, accompanied by a concurrent decrease in haemolymph clotting time. These results propose that nutritional supplementation with Vasaka leaf took no negative effect on the growth of P. monodon but did increase its non-specific immune response and can be considered as safe in shrimp culture.

Combination of Nigella sativa and Phyllanthus niruri as An Immunostimulant for The Prevention of White Spot Disease in Litopenaeus vannamei

Pacific whiteleg shrimp (Litopenaeus vannamei) is a very important commodity, which accounts for almost 70% of the total world shrimp production. However, this production is still threatened by disease attacks, one of which is caused by white spot disease (WSD). Herbs are widely used as immunostimulants as an effort to prevent diseases. Black cumin (Nigella sativa) and stone breaker (Phyllanthus niruri) have long been known as excellent immunostimulants in human. This study aimed to evaluate the potential of N. sativa and its combination with P. niruri as an immunostimulant in L. vannamei for preventing WSD. Tested shrimp (±3.0 g) were treated by N. sativa (N), P. niruri (P) and the combination of both (1N:1P, 2/3N:1/3P, 3/4N:1/4P, 1/3N:2/3, 3/5N:2/5P, 1/4N: 3/4P, 2/5N:3/5P) through feed for 28 days. They were then infected by white spot syndrome virus (WSSV) and observed for 7 days. The parameters observed included immune response and production performance. N. sativa was able to reduce the mortality of test shrimp infected with WSSV, with an RPS value of 71%. N. sativa can be combined with P. niruri in the right composition (1/2N:1/2P and 2/3N:1/3P), while the other combinations are antagonistic. The administration of the best treatments in this study, relatively did not affect the value of total hemocyte count, phenoloxidase activity, respiratory burst activity, hemolymph clotting time and production performance of tested shrimp.

Immune response and oxidative stress of shrimp Litopenaeus vannamei at different moon phases

Moon phases influence the molting process of shrimp, which affect other physiological processes as immune response. This study analyzed some parameters of immune response: total hemocytes counts (THC), hemolymph clotting time and superoxide anion (O2−) production, total protein concentration, superoxide dismutase activity, and the presence of Vibrio spp. in Litopenaeus vannamei at different moon phases. The highest percentage of organisms in intermolt stage was observed in the first quarter moon phase (95%). The highest THC was observed at new moon phase, which was significantly different (p < 0.05) than that observed at the third quarter phase. Hemolymph clotting time and CFU values of Vibrio spp. showed no significant difference (p > 0.05) between different moon phases. The higher (p < 0.05) mean O2− production value (0.400 ± 0.168 nmol min−1 mL−1) was determined in hepatopancreas at new moon phase. No relationship was observed between O2− and SOD activity, indicating that this antioxidant response was enough to counteract the influence of oxidative stress in L. vannamei at different moon phases.

Use of a methanolic olive leaf extract (Olea europaea) against white spot virus syndrome in Penaeus vannamei: Comparing the biochemical, hematological and immunological changes

The main goal of this study was to investigate the biochemical, hematological, and immunological impacts of olive (Olea europaea) leaf extract (OLE) in Penaeus vannamei experimentally infected by white spot syndrome virus (WSSV). A total of 160 juvenile whiteleg shrimp (11.1 ± 0.2 g), obtained from a private sector in Hormozgan Province (Iran), were kept for two weeks to acclimatize to conditions in the facility aquarium. The health status of the shrimp was assured by random sampling before experiment action; PCR testing confirmed the absence of common shrimp diseases. Three groups, each consisting of 20 shrimp fed with three different doses of OLE (50, 100, 200 mg/kg of the shrimp biomass), and a positive control group (not feeding with OLE), were all orally subjected to a virus challenge two weeks later. For comparison, three negative control groups with 20 shrimp were fed three different doses of OLE, and another group was fed with a diet lacking OLE. The PCR test confirmed the presence of the WSS virus in the treatment and positive control groups, while the negative control groups did not show any infections. The hemolymph of shrimp was taken on days 1, 8, and 22 from all groups after challenge for evaluation of hemolymph total protein, glucose, oxyhemocyanin (OxH), superoxide dismutase (SOD), phenoloxidase (PO) activity, coagulation time, and total hemocyte count (THC). Results showed that shrimp survival significantly increased up to 65% when fed with high doses of OLE for seven days. The maximum hemolymph total protein and glucose levels significantly belonged to the treatment that was challenged by WSSV without receiving OLE. The present findings indicate WSS infection caused a significant decrease in OxH level compared to that of the control groups. Therapeutic feeding with OLE could significantly decrease the SOD and PO activities, and the hemolymph clotting time. We did not see no remarckable effects of OLE on the total hemocyte count. In conclusion, the various indices studied in this research showed that the olive leaf extract would be useful in the control and prevention of white spot syndrome.

Long-term administration of diets containing Gracilaria tenuistipitata extract induce the expression of immune-related genes and increase the immune response and resistance against Vibrio harveyi in white shrimp Litopenaeus vannamei

White shrimp Litopenaeus vannamei juveniles (2.15 ± 0.16 g) fed diets containing Gracilaria tenuistipitata extract (GTE) at 0, 0.5, 1.0, and 2.0 g kg−1 for 16 weeks were examined for transcripts of lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PX), integrin β (IB), prophenoloxidase (proPO) I, proPO II, α-2 macroglobulin (α-2 M), cytMnSOD, mtMnSOD, glutathione peroxidase (GPx), transglutaminase (TG) I, TG II, lysozyme, and heat shock protein 70 (HSP70) as well as immune parameters. Shrimp fed the 2.0 g kg−1 GTE diet significantly exhibited up-regulated expressions of these genes, increased hyaline cells (HCs), granular cells (GCs, including semi-granular cells), total hemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, and earlier hemolymph clotting time. In a separate experiment, shrimp fed different diets for 16 weeks were challenged with Vibrio harveyi at 2 × 104 cfu shrimp−1 and then placed in seawater. The survival rate of shrimp fed the 2.0 g kg−1 GTE diet was significantly higher over 24–168 h post challenge. In conclusion, shrimp following long-term oral administration of a GTE-containing diet at 2.0 g kg−1 induced the expression of immune-related genes and exhibited increased their immune response and resistance against V. harveyi.

Dietary values of Forsythia suspensa extract in Penaeus monodon under normal rearing and Vibrio parahaemolyticus 3HP (VP3HP) challenge conditions: Effect on growth, intestinal barrier function, immune response and immune related gene expression

Two trials were conducted to determine the effects of dietary Forsythia suspensa extract (FSE) on shrimp, Penaeus monodon, first on growth performance, second on the immune response and immune related gene expression of shrimp. In trial 1, shrimp (mean initial wet weight about 3.02 g) were fed with five diets containing 0% (basal diet), 0.01%, 0.02%, 0.04% and 0.06% FSE in triplicate for 60 days. Growth performance (final body wet weight, FBW; weight gain, WG; biomass gain, BG) of shrimp fed FSE diets were higher (P < 0.05) than that of shrimp fed the basal diet. The survival among all the diets treatments were above 90% and no significant difference was revealed among them (P > 0.05). The antioxidant capacity (total antioxidant status, TAS; glutathione peroxidase, GSH-Px) appears in the trend of firstly increasing then decreasing with the increasing of dietary FSE levels. The highest value of TAS and GSH-Px were found in shrimp fed 0.02% FSE diet and were significantly higher than that of shrimp fed the basal and 0.06% FSE diets (P < 0.05). Hepatopancreas malondialdehyde (MDA) of shrimp fed FSE diets were lower (P < 0.05) than that of shrimp fed the basal diet. Total haemocyte count of shrimp fed the basal diet was lower (P < 0.05) than that of shrimp fed FSE diets. Haemolymph clotting time of shrimp had the opposite trend with the total haemocyte count of shrimp. No significant differences were found in haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) and in molecular gene expression profiles of heat shock protein 70 (Hsp 70) mRNA and hypoxia inducible factor-1α (HIF-1α) mRNA in haemolymph of shrimp among all diet treatments (P > 0.05). In trial 2, a pathogenic strain of Vibrio parahaemolyticus 3HP (VP3HP) injection challenge test was conducted for 6-day after the rearing trial and shrimp survival were also compared among treatments. Survival of shrimp fed diets supplemented with 0.01%–0.02% FSE were higher than that of shrimp fed the basal and 0.06% FSE diets (P < 0.05). Dietary FSE supplementation produced stronger hepatopancreas antioxidant capacity (TAS, GSH-Px) (P < 0.05) and higher glutathione (GSH) level (P < 0.05), lower superoxide dismutase activity (SOD) (P < 0.05), higher total haemocyte count (P < 0.05), lower haemolymph clotting time (P < 0.05), lower MDA and carbonyl protein concentration (P < 0.05), lower haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) (P < 0.05), generated lower molecular gene expression profiles of HSP 70 mRNA and higher HIF-1α mRNA (P < 0.05) than the basal diet. The immune response were characterized by lower TAS and higher antioxidant enzyme activities (SOD, GSH-Px) and higher oxidative stress level (MDA and carbonyl protein) and higher haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) compared to levels found in trail 1. However, the total haemocyte counts and haemolymph clotting times were not changed in 0.01%–0.02% FSE diets treatments between trial 1 and trial 2 (P > 0.05). The molecular gene expression profile of Hsp 70 mRNA was increased while HIF-1α mRNA was decreased when compared to trial 1. In conclusion, results suggested that dietary intake containing FSE could enhance the growth performance and antioxidant capacity of P. monodon and furthermore reduce oxidative stress and immune depression challenged by a pathogenic strain of Vibrio parahaemolyticus stress. Considering the effect of FSE on both growth performance and immune response of P. monodon, the level of FSE supplemented in the diet should be between 0.01% and 0.02%.

The hot-water extract of leaves of noni, Morinda citrifolia, promotes the immunocompetence of giant freshwater prawn, Macrobrachium rosenbergii

The hot-water Morinda citrifolia leaf extract (HMLE) was prepared for in vitro assessment on phenoloxidase (PO) activity, respiratory bursts (RBs), and phagocytic activity (PA). Furthermore, the HMLE was administrated in the diet at 0.6, 3, and 6 g (kg diet)−1 for Macrobrachium rosenbergii, and the potential effects on the immunocompetence of prawns were evaluated. PO activity, RBs, and PA in hemocytes incubated with the HMLE at 140, 20, 20, and 140 mg l−1 significantly increased. The immune parameters of the total hemocyte count (THC), differential hemocyte count (DHC), RBs, PO activity, superoxide dismutase (SOD) activity, PA, transglutaminase (TG) activity and hemolymph clotting time were evaluated before and after 1, 3, 5, 7, and 9 weeks of the feeding trial. During 9 weeks of the feeding trial, higher THCs, DHCs, RBs, PO, and TG as well as accelerated clotting times were observed in prawns fed HMLE-containing diets at 0.6 g kg−1. The mRNA expressions of prophenoloxidase, TG, crustin, and lysozyme of prawns fed HMLE-containing diets at 0.6 g kg−1 for 9 weeks of the feeding trial significantly increased. The susceptibility of prawns fed the HMLE at 0.6 g kg−1 to Lactococcus garvieae infection significantly decreased, and the relative survival percentage was 23.1%. We therefore found that HMLE administrated through the diet at 0.6 g kg−1 was capable of enhancing the immunity and resistance against L. garvieae in M. rosenbergii.

The known two types of transglutaminases regulate immune and stress responses in white shrimp, Litopenaeus vannamei

Transglutaminases (TGs) play critical roles in blood coagulation, immune responses, and other biochemical functions, which undergo post-translational remodeling such as acetylation, phosphorylation and fatty acylation. Two types of TG have been identified in white shrimp, Litopenaeus vannamei, and further investigation on their potential function was conducted by gene silencing in the present study. Total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, transglutaminase (TG) activity, haemolymph clotting time, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when shrimps were individually injected with diethyl pyrocarbonate-water (DEPC-H2O) or TG dsRNAs. In addition, haemolymph glucose and lactate, and haemocytes crustin, lysozyme, crustacean hyperglycemic hormone (CHH), transglutaminaseI (TGI), transglutaminaseII (TGII) and clotting protein (CP) mRNA expression were determined in the dsRNA injected shrimp under hypothermal stress. Results showed that TG activity, phagocytic activity and clearance efficiency were significantly decreased, but THC, hyaline cells (HCs) and haemolymph clotting time were significantly increased in the shrimp which received LvTGI dsRNA and LvTGI + LvTGII dsRNA after 3 days. However, respiratory burst per haemocyte was significantly decreased in only LvTGI + LvTGII silenced shrimp. In hypothermal stress studies, elevation of haemolymph glucose and lactate was observed in all treated groups, and were advanced in LvTGI and LvTGI + LvTGII silenced shrimp following exposure to 22 °C. LvCHH mRNA expression was significantly up-regulated, but crustin and lysozyme mRNA expressions were significantly down-regulated in LvTGI and LvTGI + LvTGII silenced shrimp; moreover, LvTGII was significantly increased, but LvTGI was significantly decreased in LvTGI silenced shrimp following exposure to 28 and 22 °C. Knockdown of LvTGI and LvTGI + LvTGII also significantly increased the mortality of L. vannamei challenged with the pathogen V. alginolyticus. The same consequences have been confirmed in LvTGII silenced shrimp in our previous study. These results indicate that LvTGI and LvTGII not only reveal a complementary effect in gene expression levels but also play a key function in the immune defence mechanism of shrimp, by regulating the haemolymph coagulation, immune parameters and immune related gene expression, and in the regulation of carbohydrate metabolism.

Humoral and haemocytic responses of Litopenaeus vannamei to Cd exposure.

White shrimp, Litopenaeus vannamei, subadults were exposed to four dilutions of the 96 h cadmium LC50 reported for postlarvae (PL12) of this species, and the effects were evaluated after 5, 48, and 96 h of exposure. While treatments did not affect survival and hemolymph clotting time increased with time, but not as a response to Cd exposure, the intensity of other responses was related to concentration, to time of exposure, and to their interaction. Hemocyanin decreased with time in all metal concentrations but increased in the control treatment, and an almost similar trend was observed with hemocyte numbers. As an initial response, phenoloxidase activity decreased with all metal concentrations, but it increased later to values similar or higher than the control treatment.

Comparison effect of dietary astaxanthin and β-carotene in the presence and absence of cholesterol supplementation on growth performance, antioxidant capacity and gene expression of Penaeus monodon under normoxia and hypoxia condition

Abstract Two trials were conducted to determine the effects of two different carotenoid sources on shrimp, Penaeus monodon , first on growth performance, secondly on the immune response of shrimp was investigated with an air emersion challenge. In trial 1, P. monodon (mean initial wet weight about 2.07 g) were fed five diets in triplicate; a basal diet (D1) without carotenoids; two diets formulated to supply 0.1% astaxanthin alone (D2), combination of 0.1% astaxanthin and 1% cholesterol (D3); two diets with 0.25% β-carotene alone (D4), combination of 0.25% β-carotene and 1% cholesterol (D5). Growth performance (final body weight, FBW; weight gain, WG; biomass gain, BG) and survival of shrimp fed D3 showed the highest values. The apparent digestibility coefficients (ADCs) of carotenoids in carotenoid-supplemented diets (D2–D5) were quietly high (> 90%), and cholesterol supplementation did not significantly improve the carotenoid ADCs any more. However, cholesterol addition significantly enhanced the tissue carotenoid retention efficiencies in astaxanthin-supplemented diet treatments (D3 vs D2) but not in β-carotene-supplemented diet treatments (D5 vs D4). Hepatopancreas malondialdehyde (MDA) and hemolymph clotting time of shrimp fed carotenoid-supplemented diets (D2–D5) were lower ( P P P > 0.05) were found in expression profiles of heat shock protein 70 (Hsp 70) mRNA and hypoxia inducible factor-1α (HIF-1α) mRNA in hepatopancreas of shrimp among all diet treatments. In trial 2, the shrimp were exposed to air during a simulated live transportation for 36 h after the rearing trial 1. No mortalities were observed in all diet treatments after 36 h of simulated live transportation. Total hemocyte counts of shrimp fed the basal and β-carotene-supplemented diets (D1, D4 and D5) significantly declined ( P P P P P. monodon , and the supplement of cholesterol could positively enhance the efficiency of astaxanthin but not β-carotene.

Suitable dietary chitosan improves the growth performance, survival and immune function of tiger shrimp,Penaeus monodon

Different levels of dietary chitosan on growth performance, survival and stress tolerance to air exposure was studied in tiger shrimp, Penaeus monodon. Shrimp (mean initial wet weight about 1.16 g) were fed with six different diets (C0, C0.05, C0.1, C0.2, C0.3 and C0.4) containing six level of chitosan (0%, 0.05%, 0.1%, 0.2%, 0.3% and 0.4% respectively) in triplicate for 60 days. Growth performance [final body wet weight (FBW); weight gain (WG); biomass gain (BG)] of shrimp fed chitosan-containing diets were higher (P 0.05) in shrimp fed C0.3 diet group. Whole body and muscle lipid contents decreased with increasing dietary chitosan levels. Plasma total cholesterol and triglyceride contents of shrimp fed C0 diet was significantly higher (P < 0.05) than that of shrimp fed chitosan-containing diets. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities of shrimp fed C0 diet were higher than those of shrimp fed chitosan-containing diets. Digestive gland malondialdehyde (MDA) and carbonyl protein contents of shrimp fed chitosan-containing diets were lower (P < 0.05) than that of shrimp fed C0 diet. Total haemocyte count of shrimp fed C0 diet was lower (P < 0.05) than that of shrimp fed chitosan-containing diets. On the contrary, the haemolymph clotting time of shrimp fed C0 diet was higher (P < 0.05) than that of shrimp fed chitosan-containing diets. In conclusion, all results suggested that dietary intake containing 0.1% and 0.2% chitosan enhanced the growth of shrimp, whereas a higher level than 0.3% and 0.4% decreased growth of shrimp. Second-degree polynomial regression analysis of WG and BG indicated that the optimum supplement of dietary chitosan level should be 0.19–0.21%.

Dietary supplementation of honeysuckle improves the growth, survival and immunity of Penaeus monodon

Two trials were conducted to determine the effects of honeysuckle on shrimp, Penaeus monodon, first on growth performance, secondly on the immune response of shrimp. In trial 1, shrimp (mean initial wet weight about 3.02 g) were fed with five diets containing 0% (basal diet), 0.1%, 0.2%, 0.4% and 0.8% honeysuckle in triplicate for 60 days. Growth performance (final body wet weight, FBW; weight gain, WG; biomass gain, BG) of shrimp fed honeysuckle diets were higher (P < 0.05) than that of shrimp fed the basal diet, shrimp fed 0.4% honeysuckle diet showed the highest value of growth performance. Shrimp fed 0.2% honeysuckle diet showed highest value of survival. The total antioxidant status (TAS) and glutathione peroxidase (GSH-Px) activity of shrimp fed 0.2%, 0.4% and 0.8% honeysuckle diets were higher (P < 0.05) than those of shrimp fed basal and 0.1% honeysuckle diets. Hepatopancreas malondialdehyde (MDA) of shrimp fed honeysuckle diets were lower (P < 0.05) than that of shrimp fed the basal diet. Total haemocyte count of shrimp fed the basal diet was lower (P < 0.05) than that of shrimp fed honeysuckle diets. Haemolymph clotting time of shrimp had the opposite trend with the total haemocyte count of shrimp. In trial 2, the shrimp were exposed to air during a simulated live transportation for 36 h after the rearing trial. The antioxidant responses were characterized by lower TAS and higher antioxidant enzyme activities (superoxide dismutase: SOD, GSH-Px) and higher oxidative stress level (MDA) in the hepatopancreas compared to levels found in trial 1. No mortalities were observed in any diet groups after 36 h of simulated live transportation. The glutathione (GSH) content and TAS of shrimp fed 0.2%, 0.4% and 0.8% honeysuckle diets were higher (P < 0.05) than those of shrimp fed the basal and 0.1% honeysuckle diets. The SOD activity of shrimp fed the basal diet was higher (P < 0.05) than that of shrimp fed honeysuckle diets. The GSH-Px activity of shrimp fed the basal diet was lower (P < 0.05) than that of shrimp fed 0.2%, 0.4% and 0.8% honeysuckle diets but without significant difference (P > 0.05) with shrimp fed 0.1% honeysuckle diet. Moreover, the oxidative stress level (MDA) recorded in the hepatopancreas with shrimp submitted to the honeysuckle diets were lower. In conclusion, results suggested that dietary intake containing honeysuckle could enhance the growth performance of P. monodon and improve its resistance to air exposure during simulated live transportation. Considering the effect of honeysuckle on both growth performance and survival of P. monodon, the level of honeysuckle supplemented in the diet should be between 0.2% and 0.4%.

Effects of Dietary Mannan Oligosaccharide on Survival, Growth, Physiological Condition, and Immunological Responses of Marron, Cherax tenuimanus (Smith 1912)

The efficacy of dietary supplementation of mannan oligosaccharide (MOS) (Bio‐Mos®, Aqua Grade; Alltech, Lexington, KY, USA) was evaluated on survival, growth, physiological and immunological parameters of marron, Cherax tenuimanus. Marron were fed six different isonitrogenous and isocalorific diets supplemented with 0, 0.05, 0.1, 0.2, 0.4, and 0.8% of MOS for a duration of 112 d and examined for survival and growth every 28 d. Physiological and immunological indicators were measured every 56 d. After 112 d, survival was the highest when marron were fed 0.2 and 0.4% MOS supplemented diets. Wet tail muscle index (TMIwet) and dry tail muscle index (TMIdry) of marron fed MOS supplemented diets were significantly higher (P &lt; 0.05) than marron fed diet without MOS. Osmoregulatory capacity was highest in marron fed 0.4% MOS. Marron fed MOS supplemented diets had lower (P &lt; 0.05) bacteraemia than marron fed diet without MOS. Hemolymph clotting time was the lowest and granular cells were the highest in marron fed 0.2 and 0.4% MOS. The results suggest that feeding 0.2–0.4% MOS in the diet increases the health status of marron.

Effects of dietary β – 1,3 – glucan on the growth, survival, physiological and immune response of marron, Cherax tenuimanus (smith, 1912)

Six isonitrogenous and isocalorific diets supplemented with five different levels of beta –1,3 – glucan (0.08%, 0.1%, 0.2%, 0.4% and 0.8%) were formulated and tested for marron ( Cherax tenuimanus) growth, survival, organosomatic indices, osmoregulatory capacity and immunological parameters (total and differential haemocyte counts, haemolymph clotting time and bacteraemia). The sixth diet without any beta – 1,3 – glucan was used as a control. Each diet was provided to 18 marron (0.47 ± 0.02 g initial weight) replicated 3 times in individual 250 L fiberglass cylindrical tanks. Each tank was provided with a biological filtration recirculating water system. After 84 days of culture, the survival and yield were higher in the marron fed 0.1% beta glucan supplemented diet. The different levels of beta glucan did not alter any of the physiological parameters of marron. However, dietary supplementation with beta glucan resulted in significantly higher ( P < 0.05) total haemocyte count (THC) and granular cells. The bacteraemia rank was lower in all diets having beta glucan supplemented with more than or equal to 0.1% compared to the control and 0.08% beta glucan supplemented diets. Results suggest that dietary beta – 1,3 – glucan at a minimum concentration of 0.1–0.2% can improve the immune system of marron.

Dietary supplementation of mannan oligosaccharide improves the immune responses and survival of marron, Cherax tenuimanus (Smith, 1912) when challenged with different stressors

Three trials were conducted to determine the effects of mannan oligosaccharide (Bio-Mos ®) on the immune responses of marron. In the first trial marron were challenged with Vibrio mimicus infection, in the second with NH 3 exposure and in the third, the marron were exposed to air during a simulated live transportation trial. For V. mimicus infection and live transportation trials, marron (10.44 ± 0.20 g and 4.44 ± 0.20 g initial weights, respectively) were fed three different diets containing 0% (control diet), 0.2% and 0.4% Bio-Mos ® for 30 days and 112 days respectively before challenge, whereas for the NH 3 exposure trial, marron (94 ± 2.17 g initial weight) were reared with the control diet and 0.4% Bio-Mos ® diet for 42 days before exposure to NH 3. Marron were examined for survival and total haemocyte count (THC), differential haemocyte count (DHC), haemolymph clotting time, bacteraemia and lysosomal membrane stability as indicators of immune responses during the course of the challenge. Survival of marron infected with bacteria and exposed to NH 3, were significantly improved when fed Bio-Mos ®. THCs were significantly reduced in marron fed the control diet when they were infected with bacteria and subjected to live transportation while it remained unchanged in the marron fed the Bio-Mos ® supplemented diets. THCs of marron fed any of the diets were reduced when they were exposed to NH 3 but the THCs were higher ( P < 0.05) in marron fed Bio-Mos ® diets. Vibrio spp. in haemolymph of marron fed the control diet significantly increased when they were infected with V. mimicus and challenged with NH 3 but it remained unchanged in the marron fed the Bio-Mos ® diets. Haemolymph clotting time was higher in marron fed the control diets when subjected to live transportation and 3 days of exposure to NH 3. After 96 h infection marron fed the Bio-Mos ® diets had longer NRR time than those fed the control diet. All the findings demonstrated the ability of Bio-Mos ® to improve the survival, health status and immunity of marron under the bacterial infection and stress conditions caused by air and NH 3 exposures.

Hemolymph clotting time as an indicator of stress in western rock lobster ( Panulirus cygnus George)

A simple method to measure clotting time was introduced in an experiment studying the effects of exercise stress on western rock lobster ( Panulirus cygnus). The treatment group lobsters ( N=8) were made to exercise (tail flipping) in a 200 l tank for 2 min. Lobsters were then sampled immediately after exercise and once again 1 h later. Controls were sampled both before the experiment started and after all the other samples were taken (pre- and post-exercise controls). Total hemocyte counts (THCs), differential hemocyte counts (DHCs), hemolymph clotting time, hemolymph protein content and lobster vigor were analyzed. THCs, DHCs, hemolymph clotting and vigor index showed response to treatment. THCs were increased significantly 1 h after exercise (6.36±0.58×10 6 cells/ml) compared to pre- and post-exercise controls (4.42±0.38 and 4.41±0.38×10 6 cells/ml, respectively). Hemolymph clotting time in the same experimental lobsters (43.7±2.0 s) were decreased compared to pre- and post-exercise control groups (56.0±4.1 and 52.7±2.2 s, respectively). Hemolymph clotting times were correlating with DHCs and hemolymph protein levels. Hemolymph clotting time is easily measured and could be a useful indicator of stress.

Effect of air exposure on the immune system of the rock lobster Panulirus cygnus

This study aimed to determine the effect of post harvest operations such as air exposure on the health status of lobsters. These effects can be assessed through measurement of selected immune parameters such as total haemocyte counts, haemolymph clotting time, bacteraemia and the differential proportion of granular cells in lobster haemolymph. Lobsters were taken from factory holding tanks and exposed to air for 2, 6, 12, and 18 h through placement in foam boxes. Controls were sampled at each time point by collecting lobsters from the same tank as the experimental animals (10 animals /treatment group) with a different group of lobsters being sampled at each time point. Air exposure caused a significant increase in clotting time at 12h and 18h. Total haemocyte counts showed a decreasing trend. The proportion of granular cells tended to be lower in air-exposed animals, the difference was significant at 2h exposure. The bacteraemia levels tended to be higher in air exposed lobsters than in controls and the difference was significant at 12h exposure. Low total haemocyte counts, high clotting times, low granular cell numbers and high bacteraemia levels implied increased susceptibility to infection and lowered immunity. The results show that air exposure has a significant adverse effect on the immune system and hence on the health status of the lobsters.