Effects of light and temperature conditions on the expression of GnRH and GtH genes and levels of plasma steroids in <Emphasis Type="Italic">Odontesthes bonariensis</Emphasis> females

In this study we examined the endocrine mediation between environmental factors (temperature and photoperiod) and the brain–pituitary–gonadal axis in females of pejerrey Odontesthes bonariensis. Changes in the expression of brain gonadotropin-releasing hormones (GnRHs) and gonadotropin (GtH) subunit [follicle stimulating-β (FSH-β), luteinizing hormone-β (LH-β), glycoprotein hormone-α (GPH-α)] genes, plasma gonadal steroids [estradiol (E₂) and testosterone (T)], gonadal histology, and gonadosomatic index (GSI) in adult females exposed to combinations of short-day (8 h) or long-day (16 h) photoperiods and low (12°C) or high (20°C) temperatures after winter conditions (8 h light, 12°C) were analyzed. Pejerrey females kept under the short photoperiod had low GSIs, and their ovaries contained only previtellogenic oocytes regardless of the experimental temperature. In contrast, females exposed to the long photoperiod had high GSIs and ovaries with vitellogenic oocytes at both temperatures. These fish also showed a significantly higher expression of sGnRH, pjGnRH, cGnRH-II (the three different GnRH variants found to date in the pejerrey brain), FSH-β, LH-β and GPH-α genes and plasma E₂ levels than those at the shorter photoperiod. No significant changes were observed in plasma T levels. Based on these results, we concluded that the increase in day length but not that of temperature triggers the maturation of pejerrey females after the winter period of gonadal rest and that this occurs by an integrated stimulation of the various components of the brain–pituitary–gonad axis.     

Effect of trawling with traditional and ‘T90’ trawl codends on fish size and on different quality parameters of cod <Emphasis Type="Italic">Gadus morhua</Emphasis> and haddock <Emphasis Type="Italic">Melanogrammus aeglefinus</Emphasis>

The effect of trawling on fish size and on different quality parameters of cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) was evaluated after conducting 16 valid hauls using two trawls in a double rig fitted with a traditional and a novel ‘T90’ codend, respectively. The total catch volume during the fishing period was 47.6 metric tons, with an average catch per codend of 1.5 (range 0.5–2.9) tons. The mean haul duration was 5 h. The catch was assessed according to fish size, mortality, external damage, initial white muscle pH and development of rigor mortis. Fillet quality (colour, blood spots, gaping) was assessed after 1 week of freeze-storage. Our results showed there was no difference between the two types of nets in terms of catch volume, but significantly slightly bigger fish were caught with T90 than with the traditional trawl net (p < 0.05). Haddock caught with the traditional trawl net had more external injuries related to the trawl gear than haddock caught with the T90 gear (p < 0.05). The gaping frequency for cod caught with the traditional trawl net tended to be higher than cod caught with the T90 gear, but the difference was not significant (p = 0.07). No other differences in fish quality between fish caught in the trawl nets were observed.     

Purification and characterization of α<Subscript>1</Subscript>-proteinase inhibitor and antithrombin III: major serpins of rainbow trout (<Emphasis Type="Italic">Oncorhynchuss mykiss</Emphasis>) and carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) blood plasma

The main serine proteinase inhibitors of rainbow trout (Oncorhynchuss mykiss) and common carp (Cyprinus carpio) blood plasma were isolated and purified. The investigated inhibitors, α₁-proteinase inhibitor (α₁-PI) and antithrombin III (AT III), act by forming stable complexes with target proteinases. The association rate constants k _on for the interaction of fish plasma inhibitors with several serine proteinases have been determined: k _on for both carp and rainbow trout α₁-PI were >10⁷ M⁻¹ s⁻¹ for human neutrophil elastase, and in the case of bovine trypsin and chymotrypsin k _on values were 2.0–5.2 × 10⁶ M⁻¹ s⁻¹. Association rate constants k _on for the interaction of carp and rainbow trout AT III with bovine trypsin and thrombin were about 1.3 × 10⁴–7.9 × 10⁵ M⁻¹ s⁻¹ without and >10⁷ M⁻¹ s⁻¹ in presence of heparin; so antithrombins require the presence of heparin to become effective proteinase inhibitors. The high degree of homology of the estimated amino acid sequences of fish inhibitors reactive site loops confirms their similarity with other proteinase inhibitors from the serpin family.     

Biochemical Composition of Seminal Plasma and Annual Variations in Semen Characteristics of Jundiá <Emphasis Type="Italic">Rhamdia quelen</Emphasis> (Quoy and Gaimard,Pimelodidae)

This study investigated the composition of milt of the South American silver catfish (Rhamdia quelen) or jundiá. The semen was taken from jundiá in different periods during the four seasons. The biochemical composition of seminal fluid and the characteristics of sperm were analyzed. The semen quantity which can be extracted per fish in one day was 0.95 ± 0.08 ml during spring (maximum) and 0.24 ± 0.03 ml during winter (minimum). Sperm density (spermatocrit) showed higher values in the spring (75.1 ± 1.3%) decreasing slightly afterwards and reaching 63.0 ± 2.4% to 65.0 ± 2.2% in the fall and winter. Immediately after water dilution, 90–100% of the spermatozoa presented vigorous straightforward motility that remained for at least 20 s. The total duration of the motility was 47.9 ± 1.3 s in the spring and 38.6 ± 0.6 s in the other seasons (P < 0.05). This pattern of motility is maintained for more than 2 h after storage of the milt at room temperature. The pH from 5 to 10 of the water dilution does not influence the sperm motility. The mean seminal pH and osmolality values were 8.7 ± 0.07 and 274.8 ± 11.2 (mOsm/kg), respectively. The ion concentration was: Na 153.7 ± 2.4, K 10.7 ± 2.4, Cl 139.4 ± 2.1, Ca 4.2 ± 0.2, Mg 0.9 ± 0.05, P 0.9 ± 0.08 (mEq/l). The total protein was 0.6 ± 0.05 mg/dl and cholesterol concentration was 13.9 ± 0.9 mg/dl.     

Age,growth and mortality estimates for populations of red snappers <Emphasis Type="Italic">Lutjanus erythropterus</Emphasis> and <Emphasis Type="Italic">L. malabaricus</Emphasis> from northern Australia and eastern Indonesia

Lutjanus erythropterus and L. malabaricus were examined for life-history differences among northern Australian and eastern Indonesian populations. Formation of opaque growth increments in otoliths began during April to September for northern Australian fishes and September to April for eastern Indonesian fishes. Maximum observed ages were greater than previously reported: 42 and 48 years for L. erythropterus and L. malabaricus, respectively. Eastern Indonesian red snappers grew faster than northern Australian fish. Growth was similar for northern Australian L. erythropterus populations. However, Kupang and Sape populations of L. erythropterus in eastern Indonesian showed different growth from the Arafura North population, which was similar to northern Australian fish. There were growth differences among northern Australian populations of L. malabaricus. Arafura South and Timor populations were similar, but differed from Groote and Weipa populations. There were no significant differences in growth among populations of L. malabaricus in eastern Indonesia. Total mortality estimates were similar between northern Australian and eastern Indonesian fish: 0.09 and 0.16 year⁻¹ for L. erythropterus and 0.11 and 0.14 year⁻¹ for L. malabaricus, respectively. Life-history characteristics of these red snappers are typical of other tropical snappers: slow-growing and long-lived with low mortality. However, population growth differences suggest that their management should be based on biological information from distinct stocks.     

Ontogenetic development of digestive enzymes and effect of starvation in miiuy croaker <Emphasis Type="Italic">Miichthys miiuy</Emphasis> larvae

The ontogenetic development of the digestive enzymes amylase, lipase, trypsin, and alkaline phosphatase and the effect of starvation in miiuy croaker Miichthys miiuy larvae were studied. The activities of these enzymes were detected prior to exogenous feeding, but their developmental patterns differed remarkably. Trypsin activity continuously increased from 2 days after hatching (dah), peaked on 20 dah, and decreased to 25 dah at weaning. Alkaline phosphatase activity oscillated at low levels within a small range after the first feeding on 3 dah. In contrast, amylase and lipase activities followed the general developmental pattern that has been characterized in fish larvae, with a succession of increases or decreases. Amylase, lipase, and trypsin activities generally started to increase or decrease at transitions from endogenous to exogenous feeding or diet changes, suggesting that these enzymatic activities can be modulated by feeding modes. The activities of all the enzymes remained stable from 25 dah onwards, coinciding with the formation of gastric glands and pyloric caecum. These results imply that specific activities of these enzymes underwent changes due to morphological and physiological modifications or diet shift during larval development but that they became stable after the development of the digestive organs and associated glands was fully completed and the organs/glands functioned. Trypsin and alkaline phosphatase were more sensitive to starvation than amylase and lipase because delayed feeding up to 2 days after mouth opening was able to adversely affect their activities. Enzyme activities did not significantly differ among feeding groups during endogenous feeding; however, all activities were remarkably reduced when delayed feeding was within 3 days after mouth opening. Initiation of larvae feeding should occur within 2 days after mouth opening so that good growth and survival can be obtained in the culture.     

PGF<Subscript>2α</Subscript> and gonadal steroid plasma levels of successful and unsuccessful spawning <Emphasis Type="Italic">Piaractus mesopotamicus</Emphasis> (Teleostei,Characiformes) females

Gonadal steroid and prostaglandin F₂α (PGF_2α) plasma levels were evaluated in successfully (SP) and unsuccessfully ovulated (UN) female Piaractus mesopotamicus. Forty-one females were injected with crude carp pituitary extract (0.6 and 5.4 mg kg^?1 with a 24-h interval between the doses) and sampled to determine the plasma concentration of 17β-estradiol (E₂), 17α-hydroxyprogesterone (17α-OHP), 17α,20β-dihydroxy-4-pregnen-3-one (DHP), PGF_2α, and testosterone (T) after each injection (first—A1 and second—A2), and at the time of ovulation for SP and UN. Two clusters were obtained using multivariate analysis: 1—composed of all A1, all A2, and some UN; and 2—composed of all SP and some UN. Median values of E₂ plasma levels were similar between clusters; however, plasma levels of T, 17α-OHP, DHP, and PGF_2α of cluster 2 (predominantly formed by SP) were higher than those of cluster 1. Since cluster 2 contained all SP and females of this cluster presented higher levels of PGF_2α, T, 17α-OHP, and DHP, here we evidently shown in an unprecedented manner that concomitant increased levels of these substances were associated with successful ovulation in this species, but such an increase was not determinant for successful ovulation due to the presence of some UN females in the same cluster 2. These findings highlight the unexplored potential of PGF_2α to be used as an accessory tool for inducing successful ovulation for fish farming purposes.     

Comparison between α-LNA and DHA in early developmental stages of <Emphasis Type="Italic">Takifugu obscurus</Emphasis> and <Emphasis Type="Italic">Takifugu rubripes</Emphasis>

ABSTRACT:   Rearing experiments were conducted to investigate the essential fatty acid requirements in the early developmental stages of river puffer Takifugu obscurus and tiger puffer T. rubripes using two n-3 series unsaturated fatty acids, α-linolenic acid (18:3n-3, α-LNA) and docosahexaenoic acid (22:6n-3, DHA), under salinity of 30 and 18.5–20.3°C. River and tiger puffer larvae used in this study were 15 and 14 days old after hatching, and their average body weights were 30.1 and 20.8 mg, respectively. The results on fatty acid requirements of these two species were evaluated from fish growth, survival, fatty acid composition of the fish body and activity test results. The DHA groups of both river and tiger puffer exhibited better survival and weight gain. However, there was no difference in the mean final body weights of river puffer between two dietary groups. Also, the DHA group of tiger puffer showed better results in the recovery test from anesthetic condition than that obtained in the LNA group. In an examination of the fatty acid compositions of the whole body, the LNA group containing no dietary DHA resulted in 0.5% DHA in tiger puffer and 1.1% DHA in river puffer . These results suggest that α-LNA from Artemia converted to eicosapentaenoic acid (20:5n-3, EPA) and to DHA successively by their fatty acid metabolism. Symptoms following essential fatty acid deficiency were not observed in any experimental groups. As river puffer did not represent a significant difference in the dietary effects between α-LNA and DHA treatment groups, its essential fatty acid requirement was assumed to be somewhat closer to that of the freshwater fishes in comparison with that of marine fishes, including tiger puffer.     

Distribution patterns of settlement-stage juveniles of <Emphasis Type="Italic">Girella punctata</Emphasis> and <Emphasis Type="Italic">Girella leonina</Emphasis> on the rocky coast of the Kanto–Izu region,Japan

The early life history of girellid fishes in Japanese waters is unclear, and little is known about their species-specific reproductive strategies. We examined seasonal changes of distribution patterns for settlement-stage juveniles of Girella punctata and Girella leonina on the rocky shore in the regions of Kanto and Izu, Japan, to infer the influence of the Kuroshio Current on their reproduction. We collected 813 settlement-stage juveniles mainly in Sagami Bay and genetically identified the species. The juveniles of G. punctata were collected on the rocky shore in Sagami Bay during April to August, with the abundant catch in May and June. Thus, we infer that juvenile G. punctata ubiquitously inhabit the rocky shore in the area in spring and summer. By contrast, juveniles of G. leonina were rarely collected in Sagami Bay, with a total catch of only 66. Notably, no juveniles were collected during the wintertime in Sagami Bay, although an abundant catch of G. leonina had been previously reported for Sagami Nada off Sagami Bay during January to March. This clear-cut difference between the areas likely reflects the difference in proximity to the path of the Kuroshio Current. We expect that the Kuroshio Current strongly influences the reproductive success of G. leonina.     

A comparison of growth and biochemical composition of <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> (Lmk.) and <Emphasis Type="Italic">Mytilus edulis</Emphasis> (L.) on the West coast of Cotentin,Normandy, France 1999–2000

The blue mussel, Mytilus edulis has been reared along the Normandy coast line since the 1960s. The gonadal cycle of this mussel species shows a sharp decrease in meat quality during the winter period after spawning. This decline in meat quality is so severe that sales have to be suspended from December to July. Another species of mussel, Mytilus galloprovincialis, which is reared in the lagoons along the French shore of the Mediterranean Sea has a different spawning cycle. An experiment was undertaken to study the meat quality of M. Galloprovincialis throughout the year after the spat were transferred from the Mediterranean Sea to the Normandy coast. This species showed an immediate adaptation to the gonadal cycle of M. edulis. Despite suggestions from researchers, no interest was expressed to consider such transfers of M. galloprovincialis in the future.     

cDNA cloning and primary structure comparison of tauropine dehydrogenase and β-alanopine dehydrogenase from the limpet <Emphasis Type="Italic">Cellana grata</Emphasis>

The primary structures of β-alanopine dehydrogenase (β-AlDH) and tauropine dehydrogenase (TaDH) from the limpet Cellana grata were determined by amino acid sequence analysis and complementary DNA (cDNA) cloning. β-AlDH and TaDH cDNAs comprised 1,479 nucleotides and 1,444 nucleotides, respectively, and both included an open reading frame of 1,206 nucleotides corresponding to 402 amino acids. The enzymes showed very high homology, with 96% amino acid identity. These enzymes were homologous to other marine invertebrate opine dehydrogenases (OpDHs), except TaDH of the marine sponge Halichondria japonica. The highest homologies were to alanopine dehydrogenase from Fusitriton oregonensis, being 57% for both enzymes. A phylogenetic tree constructed on the basis of marine invertebrate OpDHs and developed using a sequence distance method and neighbor-joining algorithm showed a tendency for the classification of animals from taxonomically derived evolutionary trees. Additionally, Cellana grata OpDHs belong to the same group as the Gastropoda OpDHs. This represents the first report concerning the primary structure of marine invertebrate β-AlDH, and primary structure comparisons of clearly different enzymes from the same species.     

Reproductive dynamics and nursery habitat preferences of two commercially important Indo-Pacific red snappers <Emphasis Type="Italic">Lutjanus erythropterus</Emphasis> and <Emphasis Type="Italic">L. malabaricus</Emphasis>

Red snappers were examined for reproductive biology and age-0 habitat preferences. Spawning in red snappers occurred throughout the year in northern Australia and eastern Indonesia; at least 10–30% of females and 40–80% of males were in ripe or spawning condition in most months. Northern Australian populations showed a spawning peak from July to December (L. erythropterus) and September to March (L. malabaricus). Eastern Indonesian L. malabaricus had a less defined pattern with two peaks: January–March and October. Size at first maturity was 240 mm for males and 250–300 mm for females. L ₅₀ estimates were similar between species in northern Australia: 270–280 mm (males) and 350–370 mm (females). Maximum batch fecundity was 676,100 oocytes for L. erythropterus and 997,000 oocytes for L. malabaricus. Higher mean abundances of age-0 L. erythropterus were found in silty and coarse sand/rubble estuarine habitats of northern Australia (456 ± 119 fish/km²) compared with sandy coastal habitats (5 ± 3 fish/km²). Most age-0 snapper caught at Sape (eastern Indonesia) were L. malabaricus (91%) with mean abundances of 312 ± 14 fish/km². The similarities in the reproductive characteristics of red snappers suggest that successful management approaches adopted in northern Australia should be considered in eastern Indonesia.     

Species differences and effects of soft coral extracts from <Emphasis Type="Italic">Sinnularia maximus</Emphasis> on the expression of cytochrome P4501A and 2N in butterflyfishes (<Emphasis Type="Italic">Chaetodon</Emphasis> spp.)

Cytochrome P450 (CYP) has been shown to confer resistance in numerous terrestrial insects that consume potentially toxic secondary metabolites in plants, but fewer studies have examined the role of critical biotransformation enzymes in allowing marine organisms to consume chemically defended foods. This study examined the expression of CYP1A and CYP2N mRNAs in several butterflyfish species, which can feed on numerous chemically defended soft and hard corals. In addition, the effect of an extract from a soft coral (Sinnularia maxima) on expression of hepatic CYP1A and CYP2 mRNAs was also examined. Fish were fed extracts on days 1, 3 and 5, and expression was examined on day 5. Phylogenetic analyses of the CYP1A cDNA from 12 species of butterflyfish (DNA, amino acid) indicate well-separated groupings according to their feeding strategies. The non-coralline feeding Chaetodon xanthurus exhibited a 7-fold higher basal expression of CYP2N8 relative to the other species studied. Although induction of CYP2N7 expression was observed in C. punctatofasciatus, CYP1A and CYP2N was largely unaffected or diminished by extract treatment in the other species of butterflyfish. These results indicated groupings of feeding strategy with CYP1A phylogeny in Chaetodon, but generally unaltered expression of CYP1A and CYP2N following dietary treatment with an extract from a chemically defended soft coral suggesting an inconclusive role of these isoforms in the detoxification of chemicals in these extracts.     

Galactooligosaccharide and a combination of yeast and β-glucan supplements enhance growth and improve intestinal condition in striped catfish <Emphasis Type="Italic">Pangasianodon hypophthalmus</Emphasis> fed soybean meal diets

The effects of galactooligosaccharide (GOS) and a combination of yeast and β-glucan (YβG) supplementation of dietary soybean meal (SBM) on the growth and digestive performance of striped catfish Pangasianodon hypophthalmus were evaluated. Four isonitrogenous (30% protein) and isocaloric (19 MJ/kg diet) diets were formulated to contain 100% fish meal (FM) protein, 55% FM protein/45% SBM protein, FM-SBM supplemented with 1% GOS, and a combination of 1% yeast and 0.1% β-glucan, respectively. Each diet was fed for 12 weeks to three groups of 30 striped catfish fingerlings (average weight 16.45?±?0.07 g) maintained in circular fiberglass tanks (600 l). Growth, feed utilization, and muscle protein composition of fish improved significantly after supplementation with either GOS or YβG compared to the unsupplemented SBM diet, but were similar to those of fish fed the FM diet. Nutrient digestibility, digestive enzyme activities, villi and microvilli length were significantly increased in fish fed the supplemented SBM diets. The gut microbiota ranking profile showed that supplementing the SBM diet with YβG and GOS gave a ranking of Verrucomicrobia, Spirochaetes, Bacteriodetes, and Actinobacteria phyla similar to that of the FM diet. Thus, diet containing 45% protein from soybean supplemented with either GOS or YβG can be recommended to improve the growth and digestive performance of striped catfish.     

Effect of starvation on activities and mRNA expression of lipoprotein lipase and hormone-sensitive lipase in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis> × <Emphasis Type="Italic">O. areus</Emphasis>)

A 4-week study was conducted to determine the effect of starvation on activities and mRNA expression of lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) in hybrid tilapia (Oreochromis niloticus × O. areus). The tissue samples were sampled once a week. Results showed that body weight (BW) and hepatosomatic index (HSI) were decreased significantly (P < 0.05) during starvation. The percentages of crude fat and crude protein in the whole body and the fat content in muscle decreased significantly (P < 0.05), while the rate of moisture and crude ash increased significantly (P < 0.05). The response of LPL, HSL activities and mRNA expression in tissues was tissue dependent. The activities of LPL and HSL in muscle at day 7 were elevated by 2.5 times (P < 0.05) and 11.8 times (P < 0.05) of the value at day 0, respectively, and both then decreased to pre-starvation levels at day 14 and finally stabilized at a certain level afterward. LPL and HSL mRNA abundance in muscle remained relatively stable between 0 and 14 day; then, a significant increase was seen after 14 days. In the liver, LPL activity maintained a significantly increasing trend during starvation, while HSL activity rose dramatically at day 7 of starvation by 2.35 times (P < 0.05) and finally stabilized at a certain level. The mRNA abundance of liver LPL increased significantly during the whole process of starvation (P < 0.05), whereas the mRNA abundance of liver HSL decreased significantly at day 7 of starvation, elevating significantly afterward (P < 0.05).     

Estimation of the spawning grounds of chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis> and spotted mackerel <Emphasis Type="Italic">Scomber australasicus</Emphasis> in the East China Sea based on catch statistics and biometric data

The spawning grounds of the chub mackerel (Scomber japonicus) and spotted mackerel (Scomber australasicus) in the East China Sea were estimated based on catch statistics of the Japanese large- and medium-type purse seine fishery from 1992 to 2006. Biometric data were obtained from specimens caught by purse seiners in the East China Sea from 1998 to 2006. Gonadosomatic index (GSI) at 50% sexual maturity of chub mackerel and spotted mackerel females was 2.5 and 2.6, respectively. Using this criterion for GSI, chub mackerel larger than 275 mm and spotted mackerel larger than 310 mm in fork length were considered to be mature. Mature chub mackerel was observed in the area of 15–22°C sea surface temperature (SST), and mature spotted mackerel was observed in the area of 17–25°C SST. The spawning period of chub mackerel ranged from February to June, and that of spotted mackerel ranged from February to May in the East China Sea. The spawning grounds were estimated from the distributions of catch per unit effort (CPUE) of spawners and SST. As a result, the spawning ground of chub mackerel was estimated to be in the central and southern part of the East China Sea and the area west of Kyushu in February, March, and April, and in the central part of the East China Sea, the area west of Kyushu and Tsushima Straight in May, and in Tsushima Straight and western part of the Sea of Japan in June. The spawning ground of spotted mackerel was estimated to be in the central and southern part of the East China Sea and southern coastal area of Kyushu in February, March, and April, and the central and southern part of the East China Sea and the area west of Kyushu in May.     

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.