Effects of non-starch polysaccharides enzyme, phytase and citric acid on activities of endogenous digestive enzymes of tilapia (Oreochromis niloticus × Oreochromis aureus)

Non-starch polysaccharides (NSPs) enzyme, phytase and citric acid have been shown to increase digestibility in pigs, poultry and some fish. To examine their roles in digestion in tilapia ( Oreochromis niloticus  ×  Oreochromis aureus ), the present study was designed to evaluate their effects on activities of a number of endogenous digestive enzymes in tilapia. Neither NSPs enzyme (1 g kg⁻¹) nor phytase (1 g kg⁻¹) affected the activities of protease in digestive organs. In contrast, NSPs enzyme increased the activities of amylase in the hepatopancreas and intestine by 11.4% and 49.5%, respectively, while phytase (1 g kg⁻¹) increased the activities of amylase in the hepatopancreas and intestine by 14.0% and 24.1%, respectively. Citric acid (10 g kg⁻¹) increased the activities of protease in stomach by 29.6%, but reduced the activities of protease in the intestine by 35.1%. Citric acid increased the activities of amylase in the hepatopancreas and intestine by 30.7% and 29.4%. Lipase activities were not affected by NSPs enzyme, phytase or citric acid. Above results suggest that NSPs enzyme, phytase and citric acid primarily influence the activities of amylase in digestive tract of tilapia.     

Thermal denaturation and autolysis profiles of myofibrillar proteins of mantle muscle of jumbo squid Docidicus gigas

ABSTRACT:    Jumbo squid was very similar to Japanese common squid in terms of myofibrillar Ca²⁺-, Mg²⁺- and K⁺(EDTA)-ATPase activities. Myofibrils of jumbo squid were significantly stabilized upon addition of Ca²⁺ and destabilized by increasing KCl concentration for heating. Incubation of muscle homogenate of jumbo squid induced a selective cleavage of myosin into two major fragments and the cleavage was inhibited by EDTA. Autolysis was prominent at and above 0.3 M NaCl where myosin filaments dissolve. The enzyme involved in the autolysis was proved to be unstable showing maximal autolysis rate at 25°C. Washing the homogenate partially reduced the autolysis activity.     

Binding characteristics of the Zn-binding membrane protein from common carp

ABSTRACT:    This study incorporated the 43 kDa Zn-binding membrane protein isolated from common carp into liposome. The specificity and strength of the binding of ⁶⁵Zn to the 43 kDa protein-liposomes, and the binding of the ⁶⁵Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that ⁶⁵Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of ⁶⁵Zn to the protein-liposomes was detected. The binding parameter of Zn to the protein was found to be: maximum binding site (N_max), 76.7 pmole/µg protein (approx. 3 mole of Zn²⁺/mole); and equilibrium dissociation constant (Kd), 0.19 µM. Of the cations introduced (Ca²⁺, Cd²⁺,Co²⁺, Cr²⁺, Cu²⁺, Fe²⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺), only Co²⁺ competed significantly with Zn. The protein-liposomes were also found to bind specifically to laminin with a N_max of 1.1 pmole/µg laminin, and Kd of 4.79 µM. No significant protein-liposome binding occurred to other extracellular matrix proteins (fibronectin, fibrinogen or vitronectin). Furthermore, the binding was specifically inhibited by the Arg-Gly-Asp (RGD) peptide or GRGDSPG, while two other analogs (GRGESPG and GRADSPG) were without effect.     

Insight into nucleotide and Ca<Superscript>2+</Superscript> binding to carp α-actin

ABSTRACT:   Nucleotides and Ca²⁺ binding to α-actin prepared from ordinary skeletal muscle of carp Cyprinus carpio was studied. When bound Ca²⁺ was removed with ethylenediaminetetraacetic acid, carp α-actin denatured more rapidly than chicken α-actin. Kinetic studies of the denaturation process showed that in the absence of divalent cations, the binding constants of ATP to carp and chicken actin were 5.0 × 10⁴/M and 1.2 × 10⁵/M, respectively. Competitive binding of Ca²⁺ between actin and 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N',N'-tetraacetic acid (Quin 2) showed that affinity of Ca²⁺ for carp actin was also lower than that for chicken actin by a factor of 1.6. These results indicated that carp actin could relatively easily denature due to the low affinities of these ligands. Enthalpy changes upon ATP binding to carp and chicken actin were −65 kJ/mol and −110 kJ/mol, respectively. Thermodynamic analyses of our results revealed that the entropy change associated with ATP binding to carp actin was significantly smaller than that to chicken actin, suggesting that structural stabilization upon ATP binding was less effective in carp actin.     

Purification and characterization of transglutaminase from squid gill

ABSTRACT: The present study used squid gill as a source of transglutaminase (TGase) because it has extremely high TGase activity compared with other tissues. The enzyme was purified using successive chromatographies of Sephacryl S-300 and hydroxyapatite columns. The yield and purification-fold of the enzymatic activity was 12.6% and 14.1-fold, respectively. The molecular mass of the purified enzyme was estimated to be 94 kDa by using sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis. Enzyme activity was enhanced 15-fold with an increase in NaCl concentration. Although the activity was dependent on Ca²⁺ concentration, it was not sufficiently activated even by 50 mM CaCl₂ in the absence of NaCl, but could be fully activated with 10 mM CaCl₂ in 0.7 M NaCl. However, in the absence of substrates, the enzyme was rapidly inactivated. The pH and temperature optima of the enzyme were approximately pH 8.0 and 20°C, respectively. It was stable in the absence of Ca²⁺ at pH 7.5–9.0 and had a rate constant (K _D ) of 1.6 × 10^–5 s^–1 for thermal inactivation at 50°C. These results in which squid gill TGase could be activated at higher concentrations of Ca²⁺ and NaCl than at a physiological concentration, suggest that contact with seawater or body fluid seems to activate the enzyme if the tissue is disrupted.     

Purification of transglutaminase from scallop striated adductor muscle and NaCl-induced inactivation

SUMMARY: Tissue type transglutaminase (TGase) was purified from scallop striated adductor muscle with successive chromatographies of DE-52 cellulose, Sephacryl S-300, and Mono Q columns. The yield and purification of the enzymatic activity was 16.6% and 101.9-fold, respectively. The molecular mass of purified enzyme was estimated to be 95 kDa by sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis. Scallop TGase was Ca²⁺-dependent and strongly inactivated by ρ-chloromercuribenzoic acid, N -ethylmaleimide, Cu²⁺, and Zn²⁺, meaning it belongs to the thiol group of enzymes as well as being a mammalian enzyme. When scallop TGase was incubated in 0.5 M NaCl without substrate for 2 h at 20°C and pH 7.5, enzymatic activity decreased to 14.4% of its original. However, a conformational change in the TGase molecule was not detected by either fluorescent, ultraviolet, and circular dichroism spectra analyses compared to the enzyme incubated without NaCl. In addition, the enzyme inactivated by NaCl was partially recovered by the dilution of salt concentration, which means that the NaCl-induced inactivation process is reversible to some extent. These results suggest that NaCl-induced modulation of the TGase molecule occurs via a small conformational change.     

Effect of water temperature on the growth performance and digestive enzyme activities of Chinese longsnout catfish (Leiocassis longirostris Günther)

The present study was carried out to investigate the influence of water temperature on the growth performance and digestive enzyme (pepsin, trypsin and lipase) activities of Chinese longsnout catfish. Triplicate groups of Chinese longsnout catfish (35.6±0.48 g, mean±SE) were reared at different water temperatures (20, 24, 28 and 32 °C). The feeding rate (FR), specific growth rate (SGR) and feed efficiency ratio (FER) were significantly affected by water temperatures and regression relationships between water temperature and FI, SGR as well as FER were expressed as FR=−0.016 T ²+0.91 T −10.88 ( n =12, R ²=0.8752), SGR=−0.026 T ²+1.39 T −17.29 ( n =12, R ²=0.7599) and FER=−0.013 T ²+0.70 T −8.43 ( n =12, R ²=0.7272). Based on these, the optimum temperatures for FR, SGR and FER were 27.66, 26.69 and 26.44 °C respectively. The specific activities of digestive enzymes at 24 or 28 °C were significantly higher than that at 20 or 32 °C. In addition, there was a significant linear regression between FR or SGR and specific activities of pepsin and lipase, which indicated that pepsin and lipase played important roles in regulating growth through nutrient digestion in Chinese longsnout catfish.     

Preparation of heavy meromyosin from the autolyzed squid mantle muscle homogenate

ABSTRACT:   Incubation of squid mantle muscle homogenate caused a selective cleavage of myosin into heavy meromyosin (HMM) and light meromyosin (LMM). HMM was isolated from the incubated homogenate by using ammonium sulfate fractionation. The purified HMM retained two types of light chain components. Its Mg²⁺-ATPase activity with or without F-actin showed a Ca-sensitivity. HMM was cleaved into subfragment-1 and subfragment-2 upon chymotryptic digestion with or without Ca²⁺, possessing different light chain composition. Two types of light chain component were kept intact when digested in the presence of Ca²⁺. Ca²⁺ stabilized HMM especially in a bound form to F-actin.     

Metabolic costs of changing the cation–anion difference in the diet of juvenile African catfish Clarias gariepinus (Burchell)

The influence of dietary cation–anion difference (CAD, Na⁺ + K⁺– Cl^–, mEq kg^–1) on energy metabolism and nitrogen losses in juvenile African catfish Clarias gariepinus (Burchell) was examined in fish exposed to different dietary CAD levels (–146, 116, 497, 713 and 828 mEq kg^–1 diet). The experiment was conducted in open circuit balance respiration chambers over a 3-week period. Five 24-h monitoring periods were carried out at 3-day intervals during the experimental period with O₂ consumption, ammonia and nitrate + nitrite (NO_x) and CO₂ production being measured at 5-min intervals for each chamber. The negative dietary CAD (–146 mEq kg^–1) resulted in the highest energy expenditures (83 kJ kg^–0.8· d^–1). With increasing dietary CAD levels, heat loss gradually decreased to minimum values of 56 kJ kg^–0.8 day^–1 at a dietary CAD level of 713 mEq kg^–1. Consequently, metabolizable energy utilization efficiency (MEU, percentage of retained energy over metabolizable energy) quadratically ( P  < 0.05) increased and reached a maximum at a dietary CAD of 713 mEq kg^–1.     

A quick method for the assessment of activity and inhibition of fish amylases

A sensitive and quick method was developed to determine the presence of α-amylase in the gut of aquatic organisms, as well as its sensitivity to inhibitors. The assay is based on the utilization of Petri dishes filled with starch–agarose gel as a substrate for the enzyme solution, which is placed in small wells punched in the surface. Circular zones produced by the action of amylase remain colourless after staining with lugol. Pure commercial porcine amylase was used to fit the better conditions for developing the assay (1 g L^–1 starch in the gels, 4 h of incubation). The diameter of the cleared zones were related to the activity of enzyme and the method detected linearly amylase activity in a range of 2–20 U well^–1, so it was used to reveal the presence of amylase in digestive extracts obtained from different sparid fish. The method was also used to evaluate the effect produced by a specific inhibitor on fish amylases, showing a linear response when the ratio inhibitor:enzyme (in units) changed from 20:1 to 2:1. Comparison of the cleared zones produced by amylases of sparid fish in the presence or absence of inhibitor, revealed differences in their sensitivity to inhibition, which ranged from 15 to 50% of total activity. The assay is proposed for a preliminary evaluation of possible inhibitors contained in feedstuffs used in fish feeding.     

A dietary energy level of 14.6 MJ kg−1 and protein-to-energy ratio of 20.2 g MJ−1 results in best growth performance and nutrient accretion in silver barb Puntius gonionotus fingerlings

Five iso-nitrogenous (300 g protein kg⁻¹ diet) and iso-lipidic (80 g kg⁻¹ diet) semi-purified experimental diets with variable energy levels of 10.5 (D-1), 12.5 (D-2), 14.6 (D-3), 16.7 (D-4) and 18.8 (D-5) MJ kg⁻¹ diets were fed to Puntius gonionotus fingerlings (average weight 1.79 ± 0.02 g) in triplicate groups (15 healthy fishes per replicate) for a period of 90 days to assess the optimum dietary energy level and protein-to-energy ratio (P/E). Fifteen flow-through cement tanks of 100 L capacity with a flow rate of 0.5 L min⁻¹ were used for rearing the fish. Maximum specific growth rate, protein efficiency ratio, protein productive value, RNA : DNA ratio, whole body protein content, digestive enzyme activity and minimum feed conversion ratio was found in fish-fed diet D-3 with 14.6 MJ kg⁻¹ energy level. There were no improvements in all these parameters with the further rise in dietary energy level. Hence, it may be concluded that the optimum dietary gross energy level for maximum growth and nutrient utilization of silver barb is 14.6 MJ kg⁻¹ diet with a resultant P/E ratio of 20.2 g protein MJ⁻¹ diet, when the dietary protein and lipid are maintained at optimum requirement levels of 300 and 80 g kg⁻¹ diet, respectively, for this species.     

Optimal dietary lipid level of silver barb, Puntius gonionotus fingerlings in relation to growth, nutrient retention and digestibility, muscle nucleic acid content and digestive enzyme activity

Five iso-nitrogenous (300 g kg⁻¹ diet) purified diets with graded level of lipid at 40 (D-1), 60 (D-2), 80 (D-3), 100 (D-4) and 120 (D-5) g kg⁻¹ diet were fed to Puntius gonionotus fingerlings for 90 days to determine their dietary lipid requirement. Two hundred and twenty-five fingerlings (average weight 2.34 ± 0.03 g) were equally distributed in five treatments in triplicate groups with 15 fish per replicate. Fifteen flow-through cement tanks of 100 L capacity with a flow rate of 0.5 L min⁻¹ were used for rearing the fish. Specific growth rate (SGR), feed conversion ratio (FCR), nutrient digestibility, retention, digestive enzyme activity, RNA : DNA ratio and whole-body composition were considered as the response parameters with respect to dietary lipid levels. Maximum SGR and minimum FCR with highest RNA : DNA ratio, whole-body protein content and digestive enzyme activity was found in D-3 group fed with 80 g kg⁻¹ diet lipid. Nutrient digestibility was similar in all the groups irrespective of the dietary lipid level. Maximum protein and energy retention was recorded at 80 g kg⁻¹ dietary lipid fed group. However, from the second-order polynomial regression analysis, the maximum growth of P. gonionotus fingerlings was found at 96.9 g lipid kg⁻¹ diet.     

Selenium nutrition of hybrid striped bass (Morone chrysops × M. saxatilis) bioavailability, toxicity and interaction with vitamin E

Two concurrent 12-week feeding trials were conducted to evaluate the bioavailability of inorganic sodium selenite and organic seleno-DL-methionine and to investigate the potential interaction between selenium and vitamin E in juvenile hybrid striped bass. In experiment 1, purified diets utilizing casein, gelatin and an amino acid premix as protein sources with a basal selenium concentration of 0.11 mg Se   kg⁻¹ were supplemented with either Na₂SeO₃ to provide selenium concentrations of 1.19, 2.00, 5.17 and 21.23 mg Se kg⁻¹ or with seleno-DL-methionine to provide 0.90, 1.26 and 2.55 mg Se kg⁻¹ and fed to juvenile hybrid striped bass in aquaria. A second experiment evaluated potential interactions by feeding these purified diets with or without supplemental vitamin E or sodium selenite, singularly or in combination. No overt selenium deficiency signs were exhibited by fish in either of the experiments; however, signs of selenium toxicity including retarded weight gain (WG), reduced feed intake and feed efficiency ratio (FER) as well as increased mortality, were observed in fish fed the diet containing more than 20 mg Se kg⁻¹. Whole-body selenium and whole-body selenium retention were linearly influenced by sodium selenite and selenomethionine. However, there was no significant effect of dietary selenium, vitamin E or their interaction on WG, FER and survival. Slope-ratio analysis showed that bioavailability of seleno-DL-methionine as a selenium source for juvenile hybrid striped bass was significantly ( P  < 0.01) higher (3.3-fold) than sodium selenite.     

Lethal effects caused by suspended particles and sediment load on zoospores and gametophytes of the brown alga <Emphasis Type="Italic">Eisenia bicyclis</Emphasis>

ABSTRACT:   The effects caused by suspended particles in seawater and sediment cover of the substrate on the attachment to the substrate of zoospores, and on the subsequent growth and survival of gametophytes of the brown alga Eisenia bicyclis were examined in the laboratory. The attachment rate to the substrate of the zoospores was remarkably reduced by the sediment cover on the substrate, and it decreased to 3.8% of the no-sediment control by a slight sediment cover of 3.0 mg/cm² (0.048 mm in thickness). The growth of gametophytes was not inhibited at 10 mg/cm² or less of sediment cover, though it was stopped at 30 mg/cm². The survival rate of gametophytes became 39.4% at 5 mg/cm² on the substrate, and gametophytes were all blighted at 30 mg/cm². The ratio of male to female of the surviving gametophytes became 81.5:18.5, when the sediment cover was 10 mg/cm². The rate of total loss ( TL [%]) of zoospores and gametophytes of Eisenia bicyclis was obtained from the following equation: TL  = 100 (1 − exp[−0.0339  C ] exp[−1.24  Q ]), where, C (mg/L) and Q (mg/cm²) indicate the concentration of suspended particles in seawater and the sediment on the substrate, respectively.     

Using Efinol®L during transportation of marbled hatchetfish, Carnegiella strigata (Günther)

The objective of this experiment was to test the efficacy of a probiotic (Efinol^®L) during transportation of marbled hatchetfish, Carnegiella strigata . Wild specimens were captured from a small stream and transported for 24 h in plastic fish boxes with a probiotic (10 mg L⁻¹) and probiotic-free water. The boxes were sampled at 3, 12 and 24 h of transport. At the end of the experiment, the survival rate was close to 100% in both treatments. Dissolved oxygen diminished with time in both treatments, but the probiotic group had significantly higher levels. Conductivity, pH and ammonia increased significantly during the transport, demonstrating higher levels in the probiotic-free group. Fish from both treatments presented very high net Na⁺ and K⁺ effluxes after 3 h of transport. At 24 h, net K⁺ effluxes in fish of the probiotic treatment reached values close to zero and a significantly lower Na⁺ efflux was observed. Cortisol levels in both treatments at 3 and 12 h were significantly higher than that in control samples. Higher body cortisol levels were observed in the probiotic-free group than that in the probiotic group at 3 and 12 h. The results demonstrate that addition of a probiotic during fish transport improves water quality and leads to fish presenting a lower stress response intensity.     

Evaluation of carbohydrate rich diets through common carp culture in manured tanks

Four diets (T₀–T₃) were formulated reducing the fishmeal (Indian) component by 100 g kg^–1 from 300 to 0 g kg^–1 and including proportionately increasing quantities of maize. Diets were fed for 120 days at 50 g kg^–1 body weight to triplicate groups of common carp (av. wt. 2.11–2.18 g) stocked at 1 m^–2 in mud bottomed cement tanks (18 m²), fertilized with poultry manure. Fish growth, SGR and FCR in the different treatments were statistically not significantly different ( P  > 0.05). PER was lowest for the 300 g fishmeal kg^–1 diet treatment (diet T₀), increasing with decrease in dietary fishmeal content (diets T₁–T₃). Fish survival ranged from 96.29 to 100%. Diets influenced carcass composition and digestive enzyme activity. A significant increase in lipid deposition was recorded with increasing dietary carbohydrate content. Amylase, protease and lipase activities were higher in fish fed with diets T₂ and T₃. The protein sparing effect of dietary carbohydrate and the economic implication of eliminating fishmeal from the diet are discussed.     

Haematological response and growth performance of Nile tilapia (Oreochromis niloticus L.) fed diets containing folic acid

Haematological response and growth performance over 150 days, and resistance to a low-temperature stress of Nile tilapia fed diets with increasing folic acid (FA) levels were evaluated. The experiment was conducted in a completely randomized design with eight FA levels (0.0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 mg kg⁻¹ feed) supplemented in purified diets (32.0% CP and 13 398 kj DE kg⁻¹). One hundred and ninety-two fingerlings were randomly assigned to 32 net cages distributed in eight 1000 L aquaria with a physical and biological filter and a temperature control system (26.0 ± 1.0 °C). For cold-induced stress, fish were transferred to 24 30 L-aquaria with individual biofilters and aeration. The water temperature was gradually reduced until it reached 13 °C. Haematological parameters evaluated before and after cold stress were total erythrocytes and leucocytes count, differential leucocyte, haemoglobin, haematocrit, total plasmatic protein and haematometric indices. Growth performance parameters were mean weight gain, feed conversion ratio and survival. Dietary FA supplementation did not influence erythropoiesis under normal temperature conditions; cold stress impaired erythropoiesis, causing hypochromic microcytic anaemia and leucopoiesis, and also neutrophilia. Growth performance is influenced by folate and supplementation between 0.5 and 1.0 mg FA kg⁻¹ diet, which makes up for nutritional demands, guaranteeing production and health under appropriate temperature conditions.     

Underwater sound detection by cephalopod statocyst

Abstract:   The cephalopod receptor of particle motion was identified. In a previous study, it was suggested that statocysts served this function, but there was no direct supporting evidence, and epidermal hair cells had not been conclusively ruled out. Experiments on Octopus ocellatus were conducted using respiratory activity as an indicator of sound perception. Intact animals clearly responded to 141-Hz particle motion at particle accelerations below 1.3 × 10⁻³ m/s², and the mean perception threshold at this frequency was approximately 6.0 × 10⁻⁴ m/s². Specimens in which the statoliths had been surgically removed did not show any response for accelerations up to 3.9 × 10⁻³ m/s² at 141 Hz, which was approximately 16 dB greater than the mean perception threshold at this frequency. Specimens that had undergone a control operation in which the statoliths remained intact showed positive responses at 2.8 × 10⁻³ m/s² for the same frequency stimulus. This indicates that the statocyst, which is morphologically similar to the inner ear system in fish, is responsible for the observed responses to particle motion in O. ocellatus . This is the first direct evidence that cephalopods detect kinetic sound components using statocysts.     

Effects of dietary phosphorus level on non-faecal phosphorus excretion from yellowtail (Seriola quinqueradiata Temminck & Schlegel) fed purified and practical diets

Non-faecal phosphorus (P) was determined for large yellowtail to estimate a minimum available P requirement (Experiment  1) and to justify inorganic P supplementation in a fish meal-based diet (Experiment 2). In Experiment 1, purified diets with incremental P concentrations were fed to yellowtail (mean weight 917 g) at a feeding rate of 1.5% of body weight. The peaks of non-faecal P excretion appeared 5–6 h after feeding in fish fed more than 4.5 g available P kg⁻¹ dry diet. Broken-line analysis indicated that the minimum available P requirement was 4.4 g kg⁻¹ dry diet. In Experiment 2, a purified diet (PR) containing 6.5 g available P kg⁻¹ and a fish meal-based diet with (F1) and without (F0) additional phosphorus were fed to yellowtail (mean weight 1.1 kg) at 1.5% (PR) and 2% (F0 and F1) feeding rates respectively. There was no significant difference in P excretion between fish fed the F0 (5.5 g soluble P kg⁻¹ dry diet) and the PR diet. However, significantly higher (34.5%) amounts of non-faecal P excretions (7.4 g soluble P kg⁻¹ dry diet) were found in fish fed F1 compared with the F0 diet. This suggested that there was an excess of dietary P in the F1 diet and that supplementation is not needed in fish meal-based diets for large yellowtail.     

Effects of oestradiol-17β or 17α-methyltestosterone administration on gonadal differentiation of largemouth bass Micropterus salmoides (Lacepède)

Monosex populations can be a valuable management tool in culture of larger size largemouth bass (>400 g). In this study, we investigated the effective mode and duration of oestrogen and androgen administrations to produce monosex largemouth bass populations. The experiment consisted of nine treatments. In oral administration groups, we fed 40-day-old fry either 200 mg of an oestradiol-17β (E₂) kg⁻¹ diet or 60 mg of a 17α-methyltestosterone (MT) kg⁻¹ diet for 30, 45 or 60 days. In bath treatments, we immersed fry in a 1 mg MT L⁻¹ solution for 5 h a day on three or six occasions. For control treatment, we fed fry an ethanol-treated diet for 45 days. The frequency of females in the control group was 53.1%. Oral administration of E₂ at all durations resulted in slight increases in the frequency of females (59.8–70.5%). Both modes of androgen administration at all durations were ineffective in altering phenotypic sex. The experimental results of our study indicated that male differentiation passed the point of being completely and functionally influenced by exogenous oestrogens, while female differentiation had already taken place and was no longer responsive to exogenous androgens in 40-day-old (33.5 mm) largemouth bass fry.