Molecular endocrinology of oocyte growth and maturation in fish

Pituitary gonadotropins (GTHs) are of primary importance in triggering oocyte growth and maturation. However, the actions of GTHs are not direct, but are mediated by the ovarian production of steroidal mediators of oocyte growth (estradiol-17β) and maturation (maturation-inducing hormone, MIH; 17α,20β-dihydroxy-4-pregnen-3-one, 17α,20β-DP in salmonid fishes; 17α,20β,21-trihydroxy-4-pregnen-3-one, 20β-S in sciaenid fishes). It is established that production of estradiol-17β and 17α,20β-DP by salmonid ovarian follicles occurs via the interaction of two cell layers, the thecal and granulosa cell layers (two-cell type model). A distinct shift in the salmonid steroidogenesis from estradiol-17β to 17α,20β-DP occurs in the ovarian follicle layer immediately prior to oocyte maturation. It is possible that this shift is a consequence of dramatic changes in the expression of the genes encoding various steroidogenic enzymes. As an initial step to address this question, we have isolated and characterized the cDNAs encoding a number of ovarian steroidogenic enzymes including the rainbow trout cholesterol side-chain cleavage cytochrome P-450, 3β-hydroxysteroid dehydrogenase (HSD), 17α-hydroxylase/17,20 lyase cytochrome P-450, aromatase cytochrome P-450 cDNAS as well as the pig 20β-HSD cDNA. Estradiol-17β stimulates the hepatic synthesis and secretion of a yolk precursor, vitellogenin. Vitellogenin is then transported to the ovary where it is selectively taken up into the oocyte by a receptor-mediated process involving specific cell-surface receptors. Estradiol-17β was also shown to induce the synthesis of egg membrane proteins in the liver. The maturation-inducing action of 17α,20β-DP and 20β-S is through the binding to the oocyte plasma membrane. This initial MIH-surface interaction is followed by the formation of the major mediator of MIH, maturation-promoting factor (MPF). We have purified MPF from mature oocytes of carp. Carp MPF consists of two components: the homolog of the cdc2⁺ gene product of fission yeast (p34^cdc2) and cyclin B. The cdc2 kinase protein is present in immature oocytes as well as in oocytes induced to mature by 17α,20β-DP treatment, while cyclin B proteins can be detected only in mature oocytes. Addition of bacterially expressed goldfish cyclin B to the extracts of immature goldfish oocytes induced MPF activation. These results suggest that the appearance of cyclin B protein is a crucial step for 17α,20β-DP-induced oocyte maturation in fish.     

Effect of a probiotic bacterium <Emphasis Type="Italic">Bacillus circulans</Emphasis> PB7 in the formulated diets: on growth,nutritional quality and immunity of <Emphasis Type="Italic">Catla catla</Emphasis> (Ham.)

Bacillus circulans PB7, isolated from the intestine of Catla catla, was evaluated for use as a probiotic supplement in the feeds for the fingerlings of Catla catla. The effect of supplement on the growth performance, feed utilization efficiency, and immune response was evaluated. Catla fingerlings (ave. wt. 6.48 ± 0.43 g) were fed diets supplemented with 2 × 10⁴ (feed C1), 2 × 10⁵ (feed C2), and 2 × 10⁶ (feed C3) B. circulans PB 7 cells per 100 g feed for 60 days at 5% of the body weight per day in two equal instalments in triplicate treatments. The control feed (CC) was not supplemented with the B. circulans. All the feeds were isocaloric and isonitrogenous. Fish fed with feed C2 displayed better growth, significantly (P ≤ 0.05) highest RNA/DNA ratio, a lower feed conversion ratio (FCR), and a higher protein efficiency ratio (PER) than the other experimental diets. Highest carcass protein and lipid was also observed in the fish fed C2 feed compared to the others. Significantly (P ≤ 0.05), highest protease was recorded in fish fed feed C2 (47.9 ± 0.016) and lowest in fish fed feed C3 (32.10 ± 0.009), where α-amylase activity did not differ significantly (P ≤ 0.05) beyond the lowest inclusion level. ALP, ACP, GOT, and GPT in the liver of Catla catla were the highest (P ≤ 0.05) in fish fed C2 feed. The highest TSP, albumin, and globulin was observed in fish treated with C2 feed after 60 days feeding trial, but the lowest glucose level was observed in the same treatment. After the feeding trial, the non-specific immunity levels and disease resistance of fish were also studied. Phagocytic ratio, phagocytic index, and leucocrit value were the highest in fish fed feed C2. After the feeding trial, the fish were challenged for 10 days by bath exposure to Aeromonas hydrophila (AH1) (10⁵c.f.u. ml⁻¹ for 1 h, and, after 7 days, 10⁷c.f.u. ml⁻¹ for 1 h). Highest survival percentage was observed in fish fed with feed C2 compared with only 6.66% in the controls, which indicated the effectiveness of B. circulans PB 7 in reducing disease caused by A. hydrophila.     

Preliminary Observations on the Reproductive Physiology of Female Orangemouth Corvina in Captivity

Seasonal changes In oocyte growth and plasma estradiol, testosterone, thyroid hormones and vitellogenin levels were monitored in three captive adult female orangemouth corvina Cynoscion xanthulus subjected to a condensed (8 mo) seasonal cycle of photoperiod and water temperature. Plasma concentrations of estradiol and testosterone began to rise when temperature increased to 28 C and photoperiod to 15 h light (midsummer conditions). This was accompanied by elevated circulating levels of vitellogenin and the appearance of vitellogenic oocytes (diameter > 100 μm). Estradiol concentrations and mean oocyte size increased concurrently during late summer conditions and were maximum during fall conditions, approximately 8 wk after the beginning of ovarian recrudescence. In contrast, plasma levels of thyroid hormones did not show any distinct seasonal changes. Gonadally recrudesced females contained several size classes of vitellogenic oocytes. Injection of these fish with a luteinizing hormone-releasing hormone analog (LHRHa) caused maturation and spawning of the largest oocytes (mean diameter of follicles × 2500 μm). Another group of vitellogenic oocytes had been recruited into this size class by 2 wk after the end of spawning, which suggests that this species is capable of repeated spawning during the reproductive season. Injection of LHRHa resulted in increased plasma levels of gonadotropin and repeated spawning over several days. LHRHa-induced final oocyte maturation, ovulation and spawning were preceded by increases in plasma levels of two teleostean maturation-inducing steroids, 17α,20β, 21-trihydroxy-4-pregnen-3-one (20β-S) and 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P₄). The results provide preliminary evidence that oocyte growth is stimulated in orangemouth corvina subjected to an abbreviated seasonal cycle in captivity under midsummer photoperiod and temperature conditions and is associated with seasonal increases in plasma estradiol concentrations and vitellogenin production. However, the relative importance of 20β-S versus 17α, 20β-P₄ in the control of final oocyte maturation in this species could not be determined from the results of the present study.     

Comparison of age estimates from scale, opercular bone, otolith, vertebrae and dorsal fin ray in Labeo rohita (Hamilton), Catla catla (Hamilton) and Channa marulius (Hamilton)

The present study was undertaken with a view to compare the precision and reliability of the age readings obtained from different bony structures of some important freshwater teleosts viz., Labeo rohita (Hamilton), Catla catla (Hamilton) and Channa marulius (Hamilton). Standard procedures were followed to prepare and study the age structures. In L. rohita and C. marulius percent agreement between reader's age estimates was highest for scales, i.e. 96.3% and 90.5%, respectively and in C. catla percent agreement was highest (93.3%) for opercular bone. When scale ages were compared with other alternative structures viz., otoliths, opercular bone, vertebral centra and dorsal fin rays, percent agreement was found highest between scale and opercular bone age estimates (77.8%) in L. rohita and between scale and otoliths (94.8%) in C. marulius. In case of C. catla highest percent agreement was found between opercular bone and scale age estimates. In L. rohita each of the ageing structure showed significant (P < 0.05) underestimation of age in comparison to scales. In C. catla mean age estimates from opercular bone were comparable (P > 0.05) to the values obtained from all other structures except dorsal fin rays. In C. marulius mean age estimates from scales were comparable (P > 0.05) to those from all other structures except from dorsal fin rays. Results indicated scales to be the most suitable structure for ageing L. rohita and C. marulius and opercular bone for C. catla. However, in C. catla also scales may be used as a non-destructive method of age estimation with satisfactory results.     

Water quality affects SDH activity, protein content and RNA:DNA ratios in fish (Catla catla, Labeo rohita and Oreochromis mossambicus) raised in ponds of a sewage-fed fish farm

The spatial and seasonal variability of the respiratory enzyme succinate dehydrogenase and the protein content were examined in different tissues of fish cultured in three ponds along the effluent gradient of a sewage-fed fish farm. Indian major carp Catla catla (150-230 g) and Labeo rohita (60-190 g) cultured in both the middle and last points of the sewage effluent (stocking pond 1) and (stocking pond 4) and Oreochromis mossambicus (50-160 g), a naturally growing fish of the inlet (facultative pond) and the out let of the sewage effluent (stocking pond 4) were procured every month during the period of January-December, 2005 and were subjected to determination of succinate dehydrogenase activity, total protein, DNA and RNA contents from gill, liver and muscle tissue respectively. The SDH activity of all three test fishes (Catla catla, Labeo rohita and Oreochromis mossambicus) was reduced significantly (ANOVA; P < 0.05) when cultured in SP-4 compared to SP-1 in the case of Catla catla and Labeo rohita and in facultative pond in the case of Oreochromis mossambicus.Conspicuous differences in the SDH activity of fish between the last and first stocking pond or the facultative pond were clearly due to the result of the differences in water quality. There was a direct relationship between SDH activity in gill tissue of any of the fish investigated and ammonia-N concentration of water or water pH. This shows that the respiratory activity of these fishes was strongly affected by the ammonia and pH of water. In other words, this suggests that as the distance from the point source increases, there was a substantial improvement of water quality in the ponds located along the sewage effluent gradient. Evidently, there is a progressive pattern of growth, survival and physiological health of fish and abundance of favorable diversity of food organisms with rich biodiversity.     

Relativein vitro effectiveness of estradiol-17β, androgens,corticosteroids, progesterone and other pregnene derivatives on germinal vesicle breakdown in oocytes of Indian major carps,Labeo rohita,Cirrhinus mrigala andCatla catla

The relative effectiveness of estradiol-17, androgens, corticosteroids, progesterone and other pregnene derivatives on germinal vesicle breakdown (GVBD) was investigatedin vitro using folliculated oocytes of three carps,Labeo rohita, Cirrhinus mrigala, andCatla catla. In all three species progesterone and 17-hydroxyprogesterone could induce GVBD but relatively 17,20-dihydroxyprogesterone was consistently found to be the most potent maturation-inducing steroid. Both estradiol-17 and testosterone were ineffective in inducing GVBD. Androsterone and dehydroepiandrosterone were found to be effective inC. catla at all the concentrations used. Deoxycorticosterone (DOC), hydrocortisone (HC) and cortisone were effective inducer of GVBD inC. catla whereas inL. rohita andC. mrigala only cortisone was found to be effective. All 5-reduced pregnenes were effective in inducing GVBD inL. rohita but inC. catla, only 5-pregnane-17-01-3,20-dione and 5-pregnane-3,17,20-triol and inC. mrigala, 5-pregnane-3-ol-20- one could induce oocyte maturation.     

Gut melatonin response to microbial infection in carp <Emphasis Type="Italic">Catla catla</Emphasis>

The purpose of present study was to demonstrate the response of gut melatoninergic system to Aeromonas hydrophila infection for 3 or 6 days and search for its correlation with the activity of different antioxidative and digestive enzymes to focus their interplay under pathophysiological conditions in carp (Catla catla). Microscopic study of gut in infected fish revealed degenerative changes in the tunica mucosa and lamina propria layers with sloughed off epithelial cells in the lumen. The activity of each digestive enzyme was reduced, but the levels of melatonin, arylalkylamine-N-acetyl transferase protein, the key regulator of melatonin biosynthesis, and different enzymatic antioxidants in gut were gradually and significantly increased with the progress of infection. Gut melatonin concentrations in A. hydrophila challenged carp by showing a positive correlation with the activity of each antioxidative enzyme, and a negative correlation with different digestive enzymes argued in favor of their functional relation, at least, during pathological stress. Moreover, parallel changes in the gut and serum melatonin titers indicated possible contribution of gut to circulating melatonin. Collectively, present carp study provided the first data to suggest that endogenous gut melatonin may be implicated to the mechanism of response to microbial infections in any fish species.     

Patterns of oocyte development in natural habitat and captive <Emphasis Type="Italic">Salminus hilarii</Emphasis> Valenciennes, 1850 (Teleostei: Characidae)

Fecundity and oocyte development in Salminus hilarii female brood stock were analyzed with the aim of investigating the impact of migration impediment on oogenesis. Histological analyses of the ovaries were performed in adult females caught in two different environments—the Tietê River (natural) and captivity—and the gonadossomatic index, oocyte diameter and fecundity determined. Five germ cell development stages (oogonium, perinucleolar, cortical alveoli, vitellogenic, ripe) and two other structures (postovulatory follicles and atretic oocytes) were observed in females caught in the river. Captive animals lacked the ripe oocytes and postovulatory follicles and had a relatively higher number of atretic oocytes. Females in captivity are known to produce larger oocytes, and they release fewer eggs in each spawn (absolute fecundity) when compared with animals that are able to migrate. Our results suggest that the Tietê River is undergoing alterations which are being reflected in the reproductive performance of S. hilarii, mainly due to the presence of atretic oocytes in females caught in the river. The lack of postovulatory follicles and ripe oocytes in captive animals reveals that migratory impediment negatively impacts final oocyte maturation. However, the stage of maturation reached is adequate for ovulation induction with hormone manipulation.     

Molecular characterization,tissue distribution of Follicle‐Stimulating Hormone (FSH) beta subunit and effect of kisspeptin‐10 on reproductive hormonal profile of Catla catla (Hamilton, 1822)

Gonadotropin (GTH) hormones are glycoprotein which stimulates gonadal maturation in vertebrates. Follicle stimulating hormone is involved in initiation of gametogenesis and regulation of gonadal growth. FSHβ has been cloned and characterized from the brain of Catla catla. The FSHβ full‐length of cDNA sequence of 523 bp comprised 3, 394 and 128 bp of 5′‐UTR, open reading frame (ORF) 3′‐UTR respectively. The coding region of C. catla FSHβ encoded a peptide of 130 amino acids. Phylogenetic analysis of C. catla FSHβ deduced amino acid sequence showed high similarity with Gobiocypris rarus followed by goldfish, Carassius auratus. The qPCR result shows that FSHβ mRNA is mainly expressed in pituitary while moderate and low expression was observed in testis and ovary respectively. Chitosan‐nanoconjugated kisspeptin‐10 (CK‐10) of particle size 125 nm, polydispersity index of 0.335 to 0.65 and zeta potential of ?34.95 mV were synthesized and evaluated at against naked kisspeptin‐10 for their reproductive hormonal profile. Treatment of fish with CK‐10 showed controlled and sustained surge of the reproductive hormones (FSH & LH) with peak at 12 h. The hormone levels of naked kisspeptin‐10 treated fish decline after 6 h. The sustained release of this CK‐10 will help in reducing maturation age, synchronization of ovulation and spawning in fish. This is the first report on use of chitosan‐nanoconjugated kisspeptin‐10 (CK‐10) for reproduction in fish.     

Dietary Arginine Requirement of Fingerling Indian Major Carp,Catla catla (Hamilton)

Dietary arginine requirement of fingerling Catla catla (3.55 ± 0.05 cm; 0.61 ± 0.02 g) was determined by feeding casein–gelatin‐based isonitrogenous (33% crude protein) and isocaloric (3.40 kcal/g digestible energy) amino acid test diets containing six graded levels of l ‐arginine (1, 1.25, 1.5, 1.75, 2, and 2.25% dry diet) for 12 wk. Maximum absolute weight gain (6.93 g/fish), protein efficiency ratio (2.13), protein deposition (0.36), arginine retention efficiency (78%), and best feed conversion ratio (1.42) were recorded in fish fed 1.75% arginine of the dry diet. Maximum carcass protein (15.57%) and RNA/DNA ratio (4.79) were also recorded for the group fed 1.75% arginine of the dry diet. Quadratic regression analysis at 95% maximum or minimum response of above growth parameters yielded optimum arginine requirement of fingerling C. catla at 1.67% of the dry diet. On the basis of the above analysis of the growth parameters, it is recommended that the inclusion of dietary arginine at 1.67% of the dry diet is optimum for formulating arginine‐balanced, cost‐effective quality feeds for the mass culture of fingerling C. catla .     

Role of photoperiod and melatonin in the regulation of ovarian functions in Indian carp <Emphasis Type="Italic">Catla catla</Emphasis>: basic information for future application

The major carp Catla catla is a popular freshwater edible fish and an extensively cultured species in India. However, basic information on the physiology of reproduction is lacking in this species. By virtue of its surface feeding habit, it maintains close contact with environmental light, and is a species of choice for the study of temporal organization of reproduction under natural and experimental conditions for understanding environmental and/or endocrine mechanisms that control the annual reproductive cycle. The present review covers information gathered in recent years to emphasize the temporal pattern of reproductive events and the importance of photoperiod and melatonin, a hormone of the pineal gland that links the environment with the endocrine system in the regulation of reproduction. Finally, the outcome of the research is discussed in terms of its direct and indirect applications to aquaculture of this species with a view to manipulate its breeding at a desired time of the year to meet market demand.     

Identification of progesterone and 17α-hydroxyprogesterone in polychaetes (Perinereis sp.) and the effects of hormone extracts on penaeid oocyte development in vitro

A possible factor behind the successful use of polychaetes for prawn broodstock diet was investigated. Some steroid hormones in polychaetes have been suspected as being a potential component for prawn reproductive system development. Progesterone (P4) and 17-alpha hydroxyprogesterone (17α-OHP4) were extracted from polychaetes (Perinereis sp.) and female Penaeus monodon broodstock at various maturation stages. Concentration of both hormones was determined using RIA or HPLC. Immature natural polychaetes (2 months old) displayed the highest level of P4 and 17α-OHP4 compared to more mature ones (4, 6 and 8 months old polychaetes) (47.48 ± 6.70 ng P4/mg Protein and 143.74 ± 28.70 ng 17α-OHP4/mg Protein) whereas opposite results were received with cultured polychaetes in which a reverse relationship between P4 and its derivative with polychaete maturation was obtained. Diverse concentrations of both hormones may be linked with other factors such as types of polychaetes, habitats, seasons, and feed intakes. Twenty-four hours in vitro incubation of prawn previtellogenic oocytes with P4 or 17α-OHP4 extracted from polychaetes or with synthetic hormones, significantly increased percentages of vitellogenic oocytes and oocytes with cortical rod compared to the control with no hormones. P4 was more effective in enhancing the final maturation of oocytes while 17α-OHP4 had more effects on vitellogenic oocytes. Synthetic steroid hormones at equal hormone concentrations produced similar results to steroid hormones extracted from natural polychaetes.     

Phytic Acid‐Induced Inhibition of Digestive Protease and α‐Amylase in Three Indian Major Carps: An in vitro Study

Antinutritional effects of phytic acid (myoinositol hexaphosphate, IP6) on growth and digestibility in fish have been reported. However, specific effect of IP6 on the digestive enzymes in fish has not been addressed. In this study, inhibitory effect of synthetic IP6 (Phytic acid sodium salt, 90% purity) on the activity of the digestive protease and α‐amylase in rohu, Labeo rohita; catla, Catla catla; and mrigal, Cirrhinus mrigala has been investigated in vitro. Graded levels (12.5, 25, 50, 100, 150, and 200 µg/mL) of IP6 were added to the reaction mixtures containing enzyme extracts and substrate solution in triplicate to detect any change in enzyme activity. Results of the experiment revealed that IP6 significantly inhibit/lower activities of the digestive enzymes in a dose‐dependent manner, as evident from the regression equations (F values significant at P < 0.001 level). Apparently, irrespective of the fish species studied IP6‐induced inhibition of α‐amylase activity was greater than protease activity. Among the three fish species studied, C. mrigala appeared to be more sensitive to IP6 for both α‐amylase and protease activity. Enzyme activity was least affected in C. catla. Results of the study might raise concern while incorporating IP6 rich plant‐derived feed ingredients in aqua feed preparation.     

Histological examination of final oocyte maturation and atresia in wild and domesticated Penaeus monodon (Fabricius) broodstock

Oocyte maturation and gonadosomatic index (GSI) of eyestalk ablated Penaeus monodon females collected from the wild and from two first‐generation domesticated lines were assessed. Frequency and diameter of the different oocytes, and the intensity of oocyte atresia, were compared among groups through histological assessments of the sections of the middle ovarian lobe. Digitized images from ovary sections were used to record the frequency and diameter of different oocyte types. Spawning performance of the three groups were expressed in terms of the percentage of females that spawned at least once (productive females), time from eyestalk ablation to first spawning (latency period) and the number of spawnings per female stocked. Final ovarian maturation was attained in all groups, as indicated by the presence of mature oocytes with cortical rods (cortical oocytes), dark‐green ovarian colour and high GSI values (5.83–6.86%). However, domesticated groups presented significant larger immature oocyte types (previtellogenic and yolky oocytes) and smaller cortical oocytes compared with wild females, indicating a reduced vitellogenic activity during final oocyte maturation. Additionally, the frequency of atresia was comparatively higher for both domesticated groups, which could be related to their inferior spawning performance. The implications of these results on the reproductive potential and development of domesticated P. monodon are discussed.     

In vitro follicle culture shows that progestagens are the maturation-inducing hormones (MIH) and possible regulators of the ovulation-mediating hormone PGE2 in female Eurasian perch Perca fluviatilis

In European aquaculture, Eurasian perch, Perca fluviatilis L., is perceived as one of the most highly valuable freshwater fish species and a strong candidate for the development of freshwater aquaculture. In the pursuit of improving the quality of reproduction in this domesticated species, investigating the hormones mediating the final oocyte maturation (FOM) is therefore indispensable. But, the exact nature of the maturation-inducing hormone (MIH) in Eurasian perch is unknown. To further validate the existence of a maturation-inducing activity behind potential hormonal candidates in this species, we in vitro tested a group of nine hormones: cortisol (Co), 11-deoxycortisol (11-D), corticosterone (coS), 11-deoxycorticosterone (DOC), 17α,20βdihydroxy-4-pregnen-3-one (DHP) and 17α,20β,21 trihydroxy-4-pregnen-3-one (THP), prostaglandin E₂ (PGE2), estradiol-17β (E2) and testosterone (T), in their ability to trigger FOM advancement and the production of sex steroids potentially involved in FOM. Using mature female perch, two in vitro experiments were conducted with oocytes at the start of the FOM. The follicles were incubated for 62 h in Cortland media with and without human chorionic gonadotropin (hCG). By the end of the incubation, only DHP and THP triggered the full advancement in FOM even at low doses with the effect of DHP being in vivo validated. However, the de novo productions of E2 and DHP were not shown to be regulated by either of the MIH candidates. Progestagens are hence more credible candidates as MIH than corticosteroids in Eurasian perch. Our in vitro study also revealed that both PGE2 and DHP are strongly associated with ovulation and that PGE2 might have slightly contributed to such DHP activity.

     

Stages of gonad development during the reproductive cycle of the blackfin snook, Centropomus medius Günther

The reproductive biology of the Blackfin snook Centropomus medius was studied to expand our current knowledge on its aquaculture potential. From April 2001 to November 2002, 301 females and 193 males were captured. A 1:1.6 (male:female) ratio was found. Gonadosomatic (GSI) and hepatosomatic (HSI) indices suggested that the spawning period (SP) extended from September to November, with peak spawning activity in September (GSI=0.98% and HSI=0.7%). The period of pre‐gametogenesis extended from January to June. During the post‐SP in December, 90% of oocytes were pre‐vitellogenic and 10% were atretic. Histological analysis of gonads (haematoxylin–eosin, Sudan black B, Alcian blue PAS, Cleveland Wolfe) showed five stages of oocyte development, each occurring at a characteristic range of oocyte diameters (μm): (I) pre‐vitellogenic (25–55); (II) cortical alveoli (56–250); (III) vitellogenic (251–340); (IV) maturation (341–645) and (V) atretic (21–100). Five reproductive stages were identified for testis morphology: (I) regressed; (II) early maturation; (III) mid‐maturation; (IV) full maturation and (V) regression. The type of ovarian development in C. medius was identified as a group synchronous.     

Influence of zinc on cadmium induced haematological and biochemical responses in a freshwater teleost fish <Emphasis Type="Italic">Catla catla</Emphasis>

The haematological (Hb, RBC, WBC) and biochemical (protein and glucose) profiles of a freshwater fish Catla catla were studied under sub-lethal toxicity of cadmium chloride for 25 days (Treatment I). In addition, the influence of zinc on cadmium toxicity was investigated for haematological and biochemical parameters (Treatment II). In both the experiments, blood haemoglobin and plasma protein level decreased in the treated fish. However, WBC, RBC and plasma glucose levels were increased in Treatment I, while Treatment II showed mixed results. In Treatment II the alterations of both haematological and biological parameters were minimal compared with Treatment I. The alterations of these parameters are discussed as non-specific biomarkers against metal toxicity in aquatic organisms.     

Dietary threonine requirement of fingerling Indian major carp,Catla catla (Hamilton) estimated by growth,protein retention efficiency,threonine deposition,haematological parameters and carcass composition

A 12‐week feeding trial was conducted to determine the dietary threonine requirement of fingerling Indian major carp, Catla catla (3.35 ± 0.11 cm; 0.59 ± 0.06 g). Six casein‐gelatin based (33% crude protein; 3.23 kcal g^?1 digestible energy) amino acid test diets with graded levels of analysed threonine (0.74%, 0.96%, 1.21%, 1.48%, 1.72% and 1.93% dry diet) were fed to satiation to triplicate groups of fish. Absolute weight gain (g per fish), feed conversion ratio, protein retention efficiency, threonine deposition, RNA/DNA ratio and carcass protein significantly improved with the increase in dietary threonine and peaked at 1.48% of the dry diet. Haematological indices were also found to be best in fish fed at 1.48% threonine diet. Quadratic regression analysis of absolute weight gain, feed conversion ratio, protein retention efficiency, threonine deposition, RNA/DNA ratio, carcass protein, haemoglobin (g dL^?1), haematocrit (%) and RBCs (10⁶ × mm^?3) at 95% of maximum and minimum response exhibited the threonine requirement of fingerling C. catla between 1.35% and 1.48% dry diet, corresponding to 4.09–4.48% dietary protein. Present finding would be useful in formulating threonine‐balanced feeds for the intensive culture of C. catla.     

Changes in oocyte and blood plasma osmotic components of ayu, Plecoglossus altivelis Temminck & Schlegel during oocyte maturation

Oocyte maturation in teleosts is characterized by a complex series of cytoplasmic and nuclear changes. The purpose of this study is to present sequential changes in the oocyte constituents and regulatory mechanisms involved in the hydration and osmotic regulation of oocytes during final maturation of ayu, Plecoglossus altivelis Temminck & Schlegel. Oocyte volumes and water contents in ayu (P. altivelis) markedly increased from 0.372 mm³ and 60.5% at the mature stage to 0.712 mm³ and 79.4% at the time of oviposition. In the same period, net increases in total free amino acids and electrolytes were noted, and involvements of these constituents in the water uptake by oocytes are discussed. Moderate oocyte hydration in ayu is accompanied by steady increases in Na⁺, but not K⁺, and these observations are in contrast to results for all other marine teleosts reported elsewhere. In the present study, a steady increase in oocyte Na⁺ during maturation until ovulation pre‐dominantly results from diffusional leakage due to concentration gradients, while at the time of oviposition, both active transport and diffusional pathways play important roles in osmotic regulation in ayu oocytes.     

Establishment and characterization of macrophage cell line from thymus of Catla catla (Hamilton, 1822)

A continuous cell line has been developed from thymus explants of Catla catla and the cells have been subcultured for 63 passages. The cells exhibited optimum growth at 30°C in L‐15 medium containing 15% foetal bovine serum. The cultured cells engulfed yeast cells and fluorescent latex beads. These cells produced reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharide and phorbol esters. The culture supernatant from the cultured cells had lysozyme activity and these cells demonstrated Fc receptors. Almost all the cells were positive for alpha‐naphthyl acetate esterase enzyme suggesting that the cells are of macrophage lineage and therefore, the cell line was designated as catla thymus macrophage (CTM) cell line. CTM cells formed aggregates around zoospores of Aphanomyces invadans, but were unable to inhibit the germination of spores. The karyotype analysis of CTM cells at 25th passage revealed a typical diploid model with 50 chromosomes per cell. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and cytochrome c oxidase subunit 1 confirmed that the CTM cell line originated from C. catla. This cell line should be useful for studying the role of macrophages in differentiation and maturation of thymocytes and can be a source of macrophage‐specific enzymes and cytokines.