Effects of dietary β‐glucan and glycyrrhizin on non‐specific immunity and disease resistance of white shrimp,Litopenaeus vannamei (Boone) challenged with Vibrio alginolyticus

The white shrimp Litopenaeus vannamei, fed immunostimulant‐free, 0.2%β‐glucan and 0.06% glycyrrhizin diets for 18 days, respectively, were challenged with Vibrio alginolyticus at 6.4 × 10⁴ CFU shrimp^?1. The total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity changes for a 120‐h period were investigated, and shrimp mortality was also recorded. The results showed that PO activity, RB and SOD activity were significantly higher in shrimp fed the two immunostimulant diets after 18 days than those in shrimp fed immunostimulant‐free diets. The THC and SOD activity decreased significantly from 0 to 24 h post challenge, and then reverted to normal levels at 96 and 72 h respectively. The values for PO activity and RB increased from 0 to 48 h post challenge. Compared with those fed the control diets, shrimp fed immunostimulants had significantly higher PO activity and RB values at 120 h post challenge. Mortalities after challenge with V. alginolyticus were significantly lower in shrimp fed with β‐glucan or glycyrrhizin than in those fed with a diet without immunostimulants. It was concluded that dietary β‐glucan and glycyrrhizin increased the shrimp immunity. Furthermore, β‐glucan caused an increase in some immune parameters 12 h earlier than glycyrrhizin after V. alginolyticus challenge.     

Immunostimulatory effects of dietary fructooligosaccharides on red swamp crayfish,Procambarus clarkii (Girard)

Fructooligosaccharides (FOS) are widely researched as prebiotics and immunostimulants in humans and terrestrial animals. However, limited researches on immune functions of FOS were reported in crustacean. In the present study, red swamp crayfish Procambarus clarkii were fed with different concentrations of FOS (2.0, 5.0, 8.0, 10.0 g kg^?1 diet), and then the immunostimulatory effects of FOS were evaluated by measuring immune parameters and examining immune‐related gene expression. PO and SOD activities were significantly increased in groups fed with 8.0 or 10.0 g kg^?1 of FOS compared with those in control group. In addition, significant elevations of SOD and phagocytic activities were also observed in group fed with 5.0 g kg^?1 of FOS. Among examined immune‐related genes, expressions of crustin1, lysozyme, SOD and pro‐PO were significantly up‐regulated by feeding with 8.0 or 10.0 g kg^?1 of dietary FOS. Survival rates of crayfish challenged with Aeromonas hydrophila were significantly improved by feeding with 5.0, 8.0 or 10.0 g kg^?1 of dietary FOS for 30 days. Phagocytic activities and bacterial clearance efficiencies were also significantly increased in groups fed with 8.0 or 10.0 g kg^?1 of FOS. The results indicated that dietary FOS effectively induced innate immune responses of red swamp crayfish in a dose‐dependent way and could be used as potential immunostimulant in crustacean.     

Dietary reduction of fish meal,supplementation of vitamin C and fish oil may promote ovary maturation by improving digestibility and immunity in Procambarus clarkii (Crustacea: Decapoda)

Fish meal, vitamin C and fish oil are known to play essential roles in reproduction and immunity in animals. To investigate how the diet affect reproduction under the condition of reducing costs, we designed an experimental diet that decreased the ratio of fish meal with soybean meal from 1:1 to 1:1.5, and supplemented with 0.06% vitamin C and 3.8% fish oil for the crayfish, Procambarus clarkii. After a 100‐day feeding experiment, female crayfish fed the test diet had significantly greater gonadosomatic index (GSI) compared with those fed the basal diet (P < 0.05). At the middle stage of ovarian development, the test diet gave significantly higher trypsin‐specific activity (340.94 ± 57.32 U mg^?1 protein) in hepatopancreas of the crayfish than the basal diet (89.48 ± 10.01 U mg^?1 protein) (P < 0.05). However, hepatosomatic index (HSI) was remarkably decreased for those females fed test diet (P < 0.05). In addition, the experimental diet resulted in markedly lower superoxide dismutase (SOD) activity and malonaldehyde (MDA) levels, especially in the ovaries (P < 0.01). These results suggest that dietary reduction of fish meal, supplementation of appropriate amounts of vitamin C and fish oil may promote ovary development probably by increasing digestibility, as well as by promoting transferring of nutrients from hepatopancreas to ovary and raising immunity of P. clarkii.     

The in vitro effects of divalent metal ions on the activities of immune‐related enzymes in coelomic fluid from the sea cucumber Apostichopus japonicus

The immune‐related enzymes in marine animals are very sensitive to divalent metal ions. To investigate the roles divalent metal ions play in the influence on the immunity of sea cucumber Apostichopus japonicus, one of the most important commercial species in Asian countries, the effects of eight divalent metal ions at concentrations of 2.5, 5, 10, 15, 20, 25 and 30 mmol L^?1 on the activities of superoxide dismutase (SOD), phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP) and myeloperoxidase (MPO) in coelomic fluid were determined with the nitro blue tetrazolium chloride (NBT) method, dopachrome formation method, p‐nitrophenyl phosphate (pNPP) method and 3,3′,5,5′‐tetramethyl benzidine (TMB) method. The results indicated that Mg²⁺ enhanced the activities of SOD, PO, ACP and AKP significantly and showed no obvious effect on MPO activity; Zn²⁺ increased the activities of SOD, ACP and AKP, and showed no obvious effect on the activities of PO and MPO; Cu²⁺ enhanced the activities of ACP, AKP and MPO and activated SOD and PO at a certain concentration range; Ca²⁺ and Mn²⁺ inhibited the activities of ACP and AKP; Fe²⁺ had strong inhibitory effect on SOD activity; Pb²⁺ showed inhibitions on the activities of SOD, PO, ACP and AKP; and Cd²⁺ inhibited MPO activity greatly. The data obtained in this study collectively suggest that Mg²⁺, Zn²⁺ and Cu²⁺ have potential in promotion of A. japonicus immunity, while Ca²⁺, Fe²⁺, Pb²⁺, Cd²⁺ and Mn²⁺ might be limiting factors to the immune response of A. japonicus.     

Immune responses of Litopenaeus vannamei to non‐ionic ammonia stress: a comparative study on shrimps in freshwater and seawater conditions

To investigate the effect of non‐ionic ammonia (NH₃‐N) stress (0.1 and 0.5 mg L^?1) on the immunity of Litopenaeus vannamei cultured in long‐term freshwater, the total haemocyte count (THC), the activity of phenoloxidase (PO), nitric oxide synthase (NOS), superoxidase dismutase (SOD) and the content of malondialdehyde (MDA) were determined and further compared with those of seawater shrimps. The results showed that NH₃‐N stress significantly reduced THC and the activity of PO and SOD (P < 0.05). Under 0.1 mg L^?1 NH₃‐N stress, NOS activity increased first and then decreased significantly, while it dropped dramatically under 0.5 mg L^?1 NH₃‐N stress (P < 0.05). During NH₃‐N stress, MDA content increased continuously, and the MDA content in hepatopancreas of freshwater shrimps was higher than that of seawater shrimps. It was concluded that NH₃‐N stress significantly influenced the non‐specific immunity and could also upset the balance of antioxidant system of L. vannamei in both freshwater and seawater shrimps. Compared with in seawater, the shrimps in freshwater were more vulnerable to NH₃‐N stress because of higher lipid peroxidation and lower immunity.     

感染溶藻弧菌对日本蟳肝胰腺免疫功能的影响

摘 要：为探讨日本蟳感染溶藻弧菌的致病机理,采用溶藻弧菌人工感染健康日本蟳,分析了感染前后日本蟳肝胰腺 PO、SOD和LSZ活性变化及注射免疫多糖后的免疫保护率,并研究了细胞超微结构的病理组织学。结果表明：日本蟳感染溶藻弧菌后,肝胰腺中PO、SOD和 LSZ的活性均受到不同程度的影响,感染6~12 h,3种酶的活性均有一个上升的过程,但之后随时间的延长呈现下降趋势;免疫感染组因感染弧菌前注射了免疫多糖,日本蟳肝胰腺中PO、SOD和LSZ酶活性均显著高于感染组的酶活性,感染7 d后的免疫保护率高达72.73％,表明免疫多糖可提高酶的活性,使机体的抗病菌能力增强。超微结构显示：对照组日本蟳肝胰腺细胞结构形态正常,上皮细胞表面的微绒毛排列整齐,各细胞器结构完整;免疫组内质网、糖原颗粒和线粒体丰富,微绒毛排列致密;感染组肝胰腺组织受到明显的损伤,细胞和部分微绒毛脱落、受损;线粒体和粗面内质网变形、水肿或仅剩一空泡;核高度异染色质化,核膜破裂。与感染组相比,免疫后感染组具有形态结构较正常的细胞核、整齐致密的微绒毛和丰富的溶酶体,但依然可见水肿的内质网和狭长畸变的线粒体。     

Changes of enzyme activity and hematopoiesis in Chinese prawn Fenneropenaeus chinensis (Osbeck) induced by white spot syndrome virus and zymosan A

White spot syndrome virus and zymosan A were used to stimulate the Chinese prawn Fenneropenaeus chinensis (Osbeck), and enzyme activities in organs involved in immune defence e.g., lymphoid organ, hepatopancreas and gill, total haemocyte count and mitotic index of haematopoietic tissue (HPT) were measured. The results showed that activities of phenoloxidase (PO), superoxide dismutase (SOD), alkaline phosphatase (ALP) and acid phosphatase (ACP) were higher in lymphoid organ than in gill. The hepatopancreas had no PO activity, its SOD activity was similar to the lymphoid organ, and its ALP and ACP activities were higher than lymphoid organ and gill. Phenoloxidase activity in lymphoid organ was higher than that of the control group at 2 h after injection and lower than control after 24 h. Superoxide dismutase activity in all organs showed an increase. Alkaline phosphatase activity in lymphoid organ and gill was higher than that of the control group at 2 and 24 h after injection respectively. The difference in ACP activity was not significant. After injection with zymosan A, the mitotic index of HPT increased by 1.5‐fold compared with the control, and returned to control level after 48 h.     

Roles of zinc and selenium in protecting sea cucumber Apostichopus japonicus coelomocytes against lipopolysaccharide‐induced oxidative stress in vitro

This study investigated the attenuate effects of zinc (Zn) and selenium (Se) on lipopolysaccharide (LPS)‐induced oxidative stress in sea cucumber Apostichopus japonicus coelomocytes in vitro. Coelomocytes were first treated with different concentrations of Zn (0.12, 0.48 and 1.2 mM) and Se (0.06, 0.24 and 0.6 mM) for 12 h and the optimal concentrations of Zn and Se as antioxidants for A. japonicus coelomocytes were selected based on antioxidant parameters including total antioxidant capacity (T‐AOC) and activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione‐S‐transferase (GST) and concentration of reduced glutathione (GSH). Next, coelomocytes were pretreated with 0 (control), 0.12 mM Zn or 0.24 mM Se for 12 h, and then were treated with LPS (100 μg mL^?1) for 6 h. Se completely inhibited LPS‐induced increase in superoxide anion () production and lipid peroxidation (LPO) and effectively prevented the LPS‐induced decreases of T‐AOC, activities of SOD, CAT, GPx and GST and concentration of GSH. Zn alleviated the LPS‐induced oxidative stress but the protective effects were not as effective as Se. The present work also proved that suboptimal amount of Zn and Se could impair the antioxidant system of A. japonicus coelomoytes. In conclusion, the present work demonstrated that both Zn and Se, especially Se, have the potentials to be the effective antioxidants for A. japonicus. Further work will be conducted for their optimal administration concentrations in vivo.     

注射白斑综合征病毒对克氏原螯虾酚氧化酶活力的影响

将白斑综合征病毒(white spot syndrome virus,WSSV)、嗜水气单胞菌(Aeromonas hydrophila,Ah)、大肠杆菌(Escherichia coli)(DH5α)用注射法接种克氏原螯虾(Procambarus clarkii),在0~72 h之间定时检测克氏原螯虾血细胞和肝胰脏中酚氧化酶(Phenoloxidase,PO)活力变化。结果显示:(1)0.1 mg/m L和1 mg/m L胰蛋白酶处理样品后,样品间差异不显著。(2)加胰蛋白酶处理与未加胰蛋白酶相比,供试克氏原螯虾PO活力均升高。(3)未加胰蛋白酶与加胰蛋白酶表现出相似的特征,WSSV和Ah注射组与对照组相比均表现为,12~48 h PO活力显著高于对照组,并且在48 h达到最大值,72 h时基本恢复正常;注射DH5α组与对照组相比没有显著性变化。可见感染WSSV后,克氏原螯虾体内酚氧化酶活力发生了变化,由此推测,PO参与了螯虾体内抵御病毒的免疫反应。     

The effects of hairtail protein hydrolysate–Fe<Superscript>2+</Superscript> complexes on growth and non-specific immune response of red swamp crayfish (<Emphasis Type="Italic">Procambarus clarkii</Emphasis>)

The experiment was conducted to investigate the effects of prepared hairtail protein hydrolysate–Fe²⁺ (PH–Fe²⁺) complexes on growth and non-specific immune responses of crayfish (Procambarus clarkii). The diets with five different levels of PH–Fe²⁺ (200, 400, 600, 800, and 1000 mg/kg) were fed to crayfish for 60 days. The results indicated that the survival rate, weight gain percentage, specific growth rate, and muscle index of crayfish with 400–1000 mg/kg of PH–Fe²⁺ feeding were significantly higher, 9.94–10.10, 8.55–8.72, 0.24–0.29 %/day, and 2.39–3.05 %, respectively, than the control values after 60 days of feeding. Additionally, after 30 days of feeding, 200–400 mg/kg of PH–Fe²⁺ showed no significant (P > 0.05) improvement effect on activities of superoxide dismutase (SOD), phenoloxidase (PO), lysozyme (LSZ), and acid phosphatase (ACP) in serum or hepatopancreas of crayfish. However, significant improvement effects were observed in 800–1000 mg/kg groups. After 60 days of feeding, 400–600 mg/kg of PH–Fe²⁺ significantly (P < 0.05) improved the activity of SOD and LSZ, but did not affect the activity of PO and ACP significantly (P > 0.05). Moreover, the activities of SOD, PO, LSZ, and ACP in serum and hepatopancreas were all significantly enhanced upon treatment with 800–1000 mg/kg of PH–Fe²⁺. For these reasons, PH–Fe²⁺ can be recommended as a supplement in crayfish feed to increase the growth and immunity.     

Direct visualization of the novel pathogen,Spiroplasma eriocheiris,in the freshwater crayfish Procambarus clarkii (Girard) using fluorescence in situ hybridization

Spiroplasma eriocheiris is the first spiroplasma strain known to be pathogenic to freshwater crustaceans. It has caused considerable economic losses both in the freshwater crayfish Procambarus clarkii (Girard) and in some other crustaceans. The monitoring of the pathogen in crustacean populations and study of its behaviour in the laboratory require the development of reliable diagnostic tools. In this article, we improved microscopic identification of S. eriocheiris by combining in situ hybridization with specific fluorescently labelled oligonucleotide probes. The established fluorescence in situ hybridization (FISH) allowed simultaneous visualization, identification and localization of S. eriocheiris in the tissues of diseased crayfish P. clarkii and exhibited low background autofluorescence and ideal signal‐to‐noise ratio. With the advantages of better tissue penetration, potentially more specific and stable, we designed three species‐specific oligonucleotide probes utilizing the sequences of 16S‐23S rRNA intergenic spacer regions (ISRs) of S. eriocheiris. Positive hybridization signals were visualized in haemocytes and connective tissues of hepatopancreas, cardiac muscle and gill from diseased crayfish. This unique distribution pattern matched the pathological changes when diagnosed by H&E staining and indicated that S. eriocheiris probably spread throughout the tissues in P. clarkii by hemokinesis. This assay will facilitate our understanding of the pathogenesis of S. eriocheiris and enhance the early diagnosis of the novel pathogen.     

Effect of dietary β‐1,3‐glucan on the immune response of Litopenaeus vannamei exposed to nitrite‐N

β‐1,3‐Glucan with 1.0 g kg^?1 was supplemented to a basal diet [control (C)] with different feeding schedules: permanently β‐1,3‐glucan diet (BG) and 14 days BG–14 days control diet (C + BG). Growth and immunological responses [respiratory burst (RB), superoxide dismutase (SOD), catalase (CAT), lysozyme and total protein] in haemolymph, hepatopancreas and muscle of Litopenaeus vannamei were recorded after 84‐day feeding and exposure to nitrite‐N (20 mg L^?1) for 120 h. β‐1,3‐Glucan administration did not affect growth performance. However, as compared with control, elevated CAT and lysozyme activities were seen in haemolymph of both BG groups, while significantly higher activities of SOD, lysozyme and RB in hepatopancreas, and higher activities of CAT and lysozyme in muscle were only seen in C + BG group. After nitrite‐N stress, significantly higher haemolymph protein, and hepatopancreas activities of lysozyme and RB were observed in both BG groups, but significantly higher activities of haemolymph SOD was only seen in C + BG group. The mortality in groups BG and C + BG was significantly lower than that in group C, but C + BG group showed a trend of higher nitrite‐N resistance compared with BG group. Considering dietary cost and immunostimulatory effects, the feeding schedule with 14 days BG–14 days control diet is more recommended.     

From fish‐processing waste to feed additives for crayfish

This research studied the in vitro compatibility of Cherax quadricarinatus digestive proteases and crude enzyme extracts recovered from fish‐processing waste to evaluate their potential use as feed additives to increase digestion efficiency in decapod crustaceans. The sources of exogenous enzymes were: Pleoticus muelleri (Pm), Artemesia longinaris (Al), and Illex argentinus (Ia). The midgut glands of early (7.2 ± 0.34 g) and advanced juveniles (13.8 ± 1.49 g) of C. quadricarinatus were used for all the assays. The compatibility and synergy between crayfish and exogenous enzymes were evaluated through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) electrophoresis and the hydrolysis of azocasein and four different meals. SDS–PAGE gels demonstrated that crayfish bands remained active when their enzymes were combined with the enzymes from Pm and Ia. An apparent enzymatic synergism was found when Pm and crayfish proteases were incubated together at pH 6 and 8 with azocasein (p ? 0.05). The ability to hydrolyze the different feed ingredients varied among the enzyme combinations assayed. Our findings suggest that fish waste has potential as a source of proteases, adding value to these processing residues and promoting a zero‐waste industry. Enzymes obtained from Pm and Ia are suitable to be used as food additives for crayfish. The results should be validated through feeding trials.     

Alteration of the haemocyte parameters,expression of immune‐related and neurohormone bursicon genes of giant river prawn Macrobrachium rosenbergii (de Man) in the response to salinity stress and ammonia stress

We examined the effects of salinity stress and ammonia stress on alteration of the haemocyte count, phenoloxidase activity, expressions of immune‐related genes including prophenoloxidase (proPO), crustin, heat shock protein 70 (HSP70), and expressions of stress‐responsive neurohormone (Bur‐α and Bur‐β) in the thoracic and abdominal ganglia of giant river prawn Macrobrachium. These parameters of prawn that subjected to salinity stress (transferred from 0‰ to 5‰ and 10‰), and subjected to ammonia‐nitrogen (ammonia‐N) stress (transferred from 0 to 0.262 mg/L and 0.786 mg/L) were examined after 0, 3, 6, 24, 72 and 168 hr respectively. During the initial period of 3, 6 and 24 hr, granulocyte haemocyte (granular and semi‐granular hemocyte) count and PO activity significantly decreased, while expressions of Bur‐α and Bur‐β significantly increased. After 24 hr, granulocyte haemocyte count and PO activity significantly increased, whereas expressions of Bur‐α and Bur‐β significantly decreased. The expressions of proPO, crustin and HSP70 were significantly downregulated in the prawn that subjected to salinity stress and ammonia‐N stress at all time periods of 3–168 hr. In conclusion, changes in the granulocyte haemocyte count of M. rosenbergii following salinity stress and ammonia‐N stress are closely associated with the changes of Bur‐α and Bur‐β expressions.     

Influence of social factors on the nocturnal activity pattern of the noble crayfish,Astacus astacus (Crustacea,Decapoda) in recirculating aquaculture systems

The social environment plays a major role in the control of growth in many crustaceans, but little is known on the mechanisms involved. Two‐ and three‐year‐old noble crayfish, Astacus astacus are strictly nocturnally active. In the present article, we studied social effects on the pattern of nocturnal activity, represented by the time which the animals spent outside shelters. For this purpose, the activity of groups of uniformly large (‘dominant’) and uniformly small (‘subordinate’) individuals were recorded at different densities both separately and in mixed set‐ups. In crowded laboratory cultures, the presence of larger (dominant) conspecifics was found to reduce the time which smaller (subordinate) individuals spent outside shelters in search of food and feeding by about one‐third. It is suggested that this asymmetric inhibitory effect is an element of the functional chain which ultimately results in an extremely wide variation in individual growth rates of communally reared A. astacus. The relevance of the findings to crayfish aquaculture is discussed.     

氨氮对拟穴青蟹的急性毒性及对其血清免疫相关酶活力的影响

采用静水法研究氨氮对拟穴青蟹(Scylla paramamosain)的急性毒性及在氨氮初始浓度分别为0(C0,对照组)、10(C10组)、20(C20组)、30(C30组)、40(C40组)、50 mg/L(C50组),胁迫时间分别为0、6、24、48、72 h的条件下对其血清中碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、溶菌酶(LZM)、超氧化物歧化酶(SOD)和酚氧化酶(PO)活力的影响。结果显示,总氨氮对拟穴青蟹24 h和48 h的半致死浓度分别为104.793、66.124 mg/L,安全浓度为7.90 mg/L,非离子氨对拟穴青蟹24 h和48 h的半致死浓度分别为8.396、5.298 mg/L,安全浓度为0.63 mg/L。在胁迫6、24、48与72 h时,各实验组的LZM活力均显著低于对照组(P0.01)。相较于对照组,C10、C20及C40组在24 h的AKP、ACP活力均显著升高(P0.05)。C20组在24 h的SOD活力则显著低于其他胁迫时间点(P0.05)。胁迫72 h时,C30、C40及C50组的PO活力显著高于对照组(P0.05)。该实验条件下,不高于40 mg/L的氨氮可在24 h内使拟穴青蟹血清中的AKP与ACP活力显著升高,而50 mg/L的氨氮则对其具有抑制作用;各实验组浓度氨氮均在72 h内对拟穴青蟹血清中的LZM活力具有显著的抑制作用,对PO活力具有明显的刺激作用,对SOD活力无显著影响。     

Evaluation of polyherbal feed supplement (Ban-V) on the enhancement of growth and non-specific immune responses in shrimp Penaeus vannamei

Owing to the growing need for eco-friendly aquaculture, usage of herbal feed supplement receives higher attention. A commercial shrimp feed supplement known as Ban-V is a unique blend of polyherbal extracts that act synergistically with each other to help in improving the immune system, protects hepatopancreas and activates several metabolic pathways for better growth and disease resistance. For that, four supplemental concentrations such as 0.5, 1.0, 2.0 and 3.0 % of Ban-V were used along with commercial shrimp feed, and feed with no supplement (control) were evaluated for 60 days. Results revealed that 2.0% of Ban-V diet fed group showed significantly increased biological performance like (average body weight, average daily growth, feed conversion ratio and survival rate) than control. The tested immunological parameters such as total haemocyte count, oxyhaemocyanin level, phenoloxidase (PO) activity, respiratory burst activity (RB), superoxide dismutase (SOD), lysozyme activity, plasma protein content (PPC) and bactericidal activity were higher in 2.0% test diet fed P. vannamei, when compared to other test and control diet fed shrimp. It is therefore suggested that Ban-V supplementation at a concentration of 2.0% was efficient in stimulating the growth and immunity in shrimp.

     

Effects of photoperiod and stocking density on survival,growth and physiological responses of narrow clawed crayfish (Astacus leptodactylus)

Photoperiod and stocking density are critical factors influencing the performance of decapod crustaceans in culture, however, their influence on growth; survival and biochemical physiology of crayfish broodstock have rarely been considered. Analysis of biochemical physiology in crayfish broodstock during the non‐breeding season provides information on the energy storage requirements of broodstock for increased survival and reproductive output. Growth rate, moulting frequency, survival and biochemical physiology were measured in Astacus leptodactylus broodstock that were cultured at three different photoperiods (18L:6D; 12L:12D; and 6L:18D) and three stocking densities (10, 20 and 40 individuals m^?2) during non‐breeding season. Survival of crayfish was highest at 18L:6D photophase and 10 m^?2 (100%) than other treatments. Survival in the high stocking density was high when combined with 18L:6D photophase, but weight gain (WG) and specific growth rate (SGR) were higher at shorter photophase and lower stocking density. Longer photophase (18L:6D) increased stress responses, characterized by increased haemolymph lactate and glucose levels. Stocking density did not affect proximate composition of crayfish; however, individuals cultured at 18L:6D photophase had higher lipid content than other photoperiod treatments. The study demonstrated that culturing A. leptodactylus at 18L:6D photophase and 10 m^?2 is critical for increased survival of broodstock.