Dietary protein modulates digestive enzyme activities and gene expression in red tilapia juveniles.

It is known that the level of dietary protein modulates the enzymatic activity of the digestive tract of fish; however, its effect at the molecular level on these enzymes and the hormones regulating appetite has not been well characterised. The objective of this study was to evaluate the effect of CP on the activity of proteases and the expression of genes related to the ingestion and protein digestion of juveniles of red tilapia (Oreochromis sp.), as well as the effects on performance, protein retention and body composition of tilapia. A total of 240 juveniles (29.32 ± 5.19 g) were used, distributed across 20 tanks of 100 l in a closed recirculation system. The fish were fed to apparent satiety for 42 days using four isoenergetic diets with different CP levels (24%, 30%, 36% and 42%). The results indicate that fish fed the 30% CP diet exhibited a higher growth performance compared to those on the 42% CP diet (P < 0.05). Feed intake in fish fed 24% and 30% CP diets was significantly higher than that in fish fed 36% and 42% CP diets (P < 0.05). A significant elevation of protein retention was observed in fish fed with 24% and 30% CP diets. Fish fed with 24% CP exhibited a significant increase in lipid deposition in the whole body. The diet with 42% CP was associated with the highest expression of pepsinogen and the lowest activity of acid protease (P < 0.05). The expression of hepatopancreatic trypsinogen increased as CP levels in the diet increased (P < 0.05) up to 36%, whereas trypsin activity showed a significant reduction with 42% CP (P < 0.05). The diet with 42% CP was associated with the lowest intestinal chymotrypsinogen expression and the lowest chymotrypsin activity (P < 0.05). α-amylase expression decreased with increasing (P < 0.05) CP levels up to 36%. No significant differences were observed in the expression of procarboxypeptidase, lipase or leptin among all the groups (P > 0.05). In addition, the diet with 42% CP resulted in a decrease (P < 0.05) in the expression of ghrelin and insulin and an increase (P < 0.05) in the expression of cholecystokinin and peptide yy. It is concluded that variation in dietary protein promoted changes in the metabolism of the red tilapia, which was reflected in proteolytic activity and expression of digestion and appetite-regulating genes.

Gene expression, enzyme activity and performance of Nile tilapia larvae fed with diets of different CP levels.

Protein is the most costly nutrient in fish feed, and while diets offered in the early stages of development typically have high levels of CP, they do not always correspond to the real requirements of the animals. Thus, research that seeks to learn the true nutritional requirements of fish is fundamental to improving commercial fish culture. The present study evaluated the protein requirements of Nile tilapia (Oreochromis niloticus) under larviculture. Fish performance, gene expression for digestive enzymes and their enzymatic activity and stress response to air exposure were analyzed. Four experimental diets differing in CP level were formulated: 30%, 36%, 42% and 48%. Fish larvae were fed the experimental diets during development and sampled 10, 20 and 30 days after the beginning of the experiment for performance, gene expression and enzymatic activity. At sampling time 30, stress resistance was also evaluated by means of an air exposure test. At sampling time 10, CP levels between 36% and 48% could be used for a better performance. During this period, pepsinogen expression was greater for 30% CP, intermediate for 42% and lower for 36% and 48%. After this initial period, diets of between 30% and 42% CP are recommended for better performance. At sampling time 20, gene expression for digestive enzymes and their enzymatic activity were similar for all diets tested. At sampling time 30, the diet of 42% CP induced both greater pepsinogen expression and pepsin activity. Survival after the air exposure test after 30 days of feeding was influenced by CP level in the diet, with the highest survival being for fish fed with 36% CP. Taken together, the present results demonstrate that dietary CP influences digestive enzyme gene expression and activity, and suggest that the best CP levels for Nile tilapia larviculture vary depending on larval stage.

The effect of environmental colour on the growth, metabolism, physiology and skin pigmentation of the carnivorous freshwater catfish Lophiosilurus alexandri.

The growth, physiology and skin pigmentation of pacamã Lophiosilurus alexandri juveniles were evaluated in an experiment using different tank colours (white, yellow, green, blue, brown and black) over an 80 day period. The tank colours did not cause significant differences to final body mass, total length, survival rate, carcass composition (moisture, crude protein, ash, ether extract, calcium, phosphorus, energy), or to plasma protein, triglyceride and cholesterol values. Haematocrit values, however, were highest for fish kept in white tanks (ANOVA P < 0·05), while the greatest haemoglobin levels were recorded for fish kept in blue and brown tanks (P < 0·01). The concentrations of cortisol (P < 0·001) and glucose (P < 0·01) were the most in fish in the black tanks. Tank colour affected skin pigmentation significantly, with fish in white tanks having the highest values of L* (brightness) and the lowest values in blue and black tanks. L*, however, decreased in all treatments throughout the experiment. C*ab increased significantly over the course of the experiment in fish kept in white tanks. Similar increases of C*ab were recorded in the other treatments but to a lesser extent. The use of black tanks during the cultivation of L. alexandri caused stress and should be avoided. Cultivation in white and yellow tanks produced individuals with a pale skin colour, while cultivation in blue and black tanks resulted in juveniles with a darker and more pigmented skin.

Effects of supplemental dietary vitamin C on quality of semen from Nile tilapia (Oreochromis niloticus) breeders.

The objective of this study was to evaluate the effects of vitamin C on growth and quality of semen from Oreochromis niloticus breeders. One hundred and sixty males were fed with different levels of vitamin C (0, 261, 599 and 942 mg/kg diet). The higher weight values were recorded for 599 (166 g) and 942 (175 g) mg of vitamin C/kg diet. Sperm motility, vigour and concentration were higher with 599 and 942 mg of vitamin C/kg diet. The semen volume, gonadosomatic index and plasma protein data from the last week showed a direct relationship with increasing levels of vitamin C. No changes were observed in the hepatosomatic index and blood glucose. The haematocrit and erythrocyte showed higher values estimated by equations derived at 850 and 638 mg vitamin C/kg diet, respectively. The leucocytes were inversely proportional to the increasing levels of vitamin C. After 100 days of feeding, animals fed the diet containing 942 mg vitamin C/kg diet had higher sperm motility, linearity, curvilinear velocity, straight line velocity and average path velocity (p < .05). Higher values of beat cross-frequency were observed in broodfish fed diets containing 942 and 599 mg vitamin C/kg. The different vitamin C levels did not cause differences in straightness, lateral head displacement and sperm morphology. For Nile tilapia males on intensive rearing and handling conditions, vitamin C levels between 599 and 942 mg/kg may be used for a better performance and quality of semen.

New hosts and genetic diversity of Flavobacterium columnare isolated from Brazilian native species and Nile tilapia.

Flavobacterium columnare is responsible for disease outbreaks in freshwater fish farms. Several Brazilian native fish have been commercially exploited or studied for aquaculture purposes, including Amazon catfish Leiarius marmoratus × Pseudoplatystoma fasciatum and pacamã Lophiosilurus alexandri. This study aimed to identify the aetiology of disease outbreaks in Amazon catfish and pacamã hatcheries and to address the genetic diversity of F. columnare isolates obtained from diseased fish. Two outbreaks in Amazon catfish and pacamã hatcheries took place in 2010 and 2011. Four F. columnare strains were isolated from these fish and identified by PCR. The disease was successfully reproduced under experimental conditions for both fish species, fulfilling Koch's postulates. The genomovar of these 4 isolates and of an additional 11 isolates from Nile tilapia Oreochromis niloticus was determined by 16S rRNA restriction fragment length polymorphism PCR. The genetic diversity was evaluated by phylogenetic analysis of the 16S rRNA gene and repetitive extragenic palindromic PCR (REP-PCR). Most isolates (n = 13) belonged to genomovar II; the remaining 2 isolates (both from Nile tilapia) were assigned to genomovar I. Phylogenetic analysis and REP-PCR were able to demonstrate intragenomovar diversity. This is the first report of columnaris in Brazilian native Amazon catfish and pacamã. The Brazilian F. columnare isolates showed moderate diversity, and REP-PCR was demonstrated to be a feasible method to evaluate genetic variability in this bacterium.

Salinity and temperature variations reflecting on cellular PCNA, IGF-I and II expressions, body growth and muscle cellularity of a freshwater fish larvae.

The present study assessed the influence of salinity and temperature on body growth and on muscle cellularity of Lophiosilurus alexaxdri vitelinic larvae. Slightly salted environments negatively influenced body growth of freshwater fish larvae and we observed that those conditions notably act as an environmental influencer on muscle growth and on local expression of hypertrophia and hypeplasia markers (IGFs and PCNA). Furthermore, we could see that salinity tolerance for NaCl 4gl(-)(1) diminishes with increasing temperature, evidenced by variation in body and muscle growth, and by irregular morphology of the lateral skeletal muscle of larvae. We saw that an increase of both PCNA and autocrine IGF-II are correlated to an increase in fibre numbers and fibre diameter as the temperature increases and salinity diminishes. On the other hand, autocrine IGF-I follows the opposite way to the other biological parameters assessed, increasing as salinity increases and temperature diminishes, showing that this protein did not participate in muscle cellularity, but participating in molecular/cellular repair. Therefore, slightly salted environments may provide adverse conditions that cause some obstacles to somatic growth of this species, suggesting some osmotic expenditure with a salinity increment.

Influência do tempo de transporte para juvenis de pacamã (Lophiosilurus alexandri) / Influence of transport time on pacama juveniles (Lophiosilurus alexandri)

The aim of this work was to evaluate different transport times for pacama juvenile (Lophiosilurus alexandri) previously trained to feed formulated diets. Four hundred and eighty-six pacama juveniles (2.52±0.71g) were submitted to 24 hours of fasting before transport. The animals were divided into nine plastic bags, with density of 54 juveniles in 5L of clean water for each bag after that inflated with pure oxygen and then sealed. Fish were transported for three hours and bags were open at 5, 6 and 7.5 hours after closing. For each time three replications were used. Survival was monitored for 96 hours post-transport. The longest time tested showed the lowest values of dissolved oxygen and pH, while the total ammonia was similar in the different treatments. Survival was 100% for different transport times after 96 hours. Some juveniles returned to feed 48 hours and the majority 72 hours after opening the bags, suggesting the efficiency of transport made at different times.

Performance and nutrient utilization in South American juvenile catfish Pseudoplatystoma spp. weighting 89 - 170g, fed at different energy and protein levels / Desempenho e utilização de nutrientes em juvenis de surubim Pseudoplatystoma spp. alimentados com diferentes níveis de energia e proteína

Most studies in nutrition for the South American catfish (surubim) were limited to the initial phase of development. However, it is clear that performance and nutrient utilization can change during the life stages of a fish. Therefore, this study aimed to evaluate the performance and nutrient utilization in juveniles of surubim fed diets varying in protein and energy levels. Two experiments were performed to test different levels of energy and protein in formulated diets. In the first experiment, surubim juveniles (89.2±4.8g) were fed five diets containing different levels of energy (18.0, 18.8, 19.6, 20.5, 21.3 MJ/kg). In the second experiment, juveniles (170.03±3.35g) were fed five diets containing different levels of protein (360, 400, 440, 480 and 520g/kg). The most favorable energy level for weight gain was 20.3 MJ/kg. The increasing energy levels provided a rise in fat and decrease in protein whole-body composition. The protein amount was between 360 to 400g/kg (383g/kg), which was adequate for performance and nutrient assimilation in surubim juveniles.

A maior parte dos estudos a respeito dos aspectos nutricionais do surubim está limitada às primeiras fases de desenvolvimento. Entretanto, é claro que o desempenho e a utilização dos nutrientes podem mudar durante os diferentes estágios de desenvolvimento destes animais. Assim sendo, este estudo teve como objetivo avaliar o desempenho e a utilização de nutrientes em juvenis de surubim alimentados com dietas contendo níveis variáveis de energia e proteína. Dois experimentos foram realizados para testar os diferentes níveis de proteína e energia. No primeiro experimento, juvenis de surubim (89,2±4,8g) foram alimentados com cinco dietas contendo níveis diferentes de energia (18.0, 18.8, 19.6, 20.5, 21.3MJ/kg). No segundo experimento, os juvenis (170,03±3,35g) foram alimentados com dietas contendo cinco níveis de proteína (360, 400, 440, 480 e 520g/kg). O melhor nível de energia para ganho de peso foi 20,3 MJ/kg. O aumento dos níveis de energia levou a um incremento nos níveis de lipídeo e diminuição da proteína corporal. Níveis de proteína entre 360 a 400g/kg foram os mais adequados para o desempenho e utilização dos nutrientes em juvenis grandes de surubim.

Production of oocytes of Nile tilapia (Oreochromis niloticus) for in vitro fertilization via hormonal treatments.

Only a few studies have described hormonal treatments for induction of synchronicity and gamete collection in Nile tilapia (Oreochromis niloticus), both important for assortative matings in breeding programmes and essential for polyploidy technologies. In this study, we compared the effectiveness of carp pituitary extract (CPE), Nile tilapia pituitary extract (TPE), human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone (GnRH) protocols on the induction of spawning and egg production in Nile tilapia. Among the hormonal treatments analysed, only hCG was effective for producing viable gametes for in vitro fertilization. To verify the viability of this hormonal treatment, hCG was tested using different doses (1000, 2000, 3000, 4000 and 5000 IU/kg) in a large number of females (208 animals) from two Nile tilapia lines. The results indicated that hCG doses between 1000 and 5000 IU/kg could be used to induce final oocyte maturation in Nile tilapia with collection of stripped oocytes. This is the first study to report differential reproductive responses to hormonal treatment between tilapia lines: line 1 was more efficient at producing eggs and post-hatching larvae after hCG induction than line 2. In conclusion, we demonstrated that the hCG protocol may be applied on a large scale to induce final oocyte maturation in Nile tilapia. The development of a protocol for in vitro fertilization in Nile tilapia may aid in breeding programmes and biotechnological assays for the development of genetically modified lines of Nile tilapia.

Produção do híbrido "cachadia" em diferentes densidades de estocagem em sistema de recirculação de água / Production of cachadia hybrid at different densities in a recirculating aquaculture system

Avaliou-se o efeito da densidade de estocagem na produção de "cachadia" (Pseudoplatystoma fasciatum x Leiarius marmoratus) em sistema de recirculação de água. Nas duas primeiras fases, com duração de 27 e 40 dias e usando animais com média de peso inicial de 31 e 57g, respectivamente, foram testados 20, 40, 60 e 80 peixes/m³. Na terceira e quarta fase, usando peixes com média de peso de 169 e 399g durante 41 e 99 dias, respectivamente, foram testados 10, 20, 30 e 40 peixes/m³. A taxa de sobrevivência foi acima de 92 por cento, sem diferenças significativas entre tratamentos. Verificou-se redução no ganho de peso diário com redução com o incremento da densidade na segunda e na quarta fase experimental. A biomassa final apresentou relação direta com a densidade em todas as fases. Taxas de conversão alimentar entre 0,8 e 1,3:1 foram registradas. O "cachadia" atingiu 1,1kg em 207 dias de cultivo, mostrando-se promissor para a piscicultura intensiva.

The aim of this study was to investigate the effect of different stocking densities on cachadia production (Pseudoplatystoma fasciatum x Leiarius marmoratus) in recirculating aquaculture system. In the two first phases for 27 and 40 days using fish with average weight of 31 and 57g, respectively, densities 20, 40, 60, and 80 fish/m³ were tested. In the third and fourth trial, using fish with an average weight of 169 and 399g during 41 and 99 days, respectively, densities 10, 20, 30, and 40 fish/m³ were tested. Survival rate was higher than 92 percent, without stocking density differences between the treatments. Daily weight gain showed reduction with the increase of the density in the second and fourth trial. However, final biomass presented direct relationship with increase density in all trials. Feed conversion rates ranged 0.8 from 1.3:1 were registered. Cachadia hybrid reached 1.1kg in approximately seven months of rearing indicating promise for intensive production.

Effect of water flow rate and feed training on "pacamã" (Siluriforme: Pseudopimelodidae) juvenile production / Efeito do fluxo de água e do condicionamento alimentar na produção de juvenis de pacamã (Siluriforme: Pseudopimelodidae)

The effects of different water flow rates and feed training on the production of "pacamã" Lophiosilurus alexandri juveniles were evaluated. In the first experiment, nine day post-hatch larvae (n= 2,400) were stocked at a density of 5 larvae/L. Different water flow (F) rates were tested: F1 = 180; F2 = 600; F3 = 1,300; and F4 = 2,600mL/min. Artemia nauplii were offered as food during the first 15 days of active feeding. In the second experiment for feed training, 720 juveniles (total length of 22.2mm) were stocked at a density of 1.5 juveniles/L. A water flow rate similar to F1 was used. The use of extruded dry diet was tested, and feed training was done with and without other enhanced flavors (Artemia nauplii or Scott emulsion). The water flow rates did not influence the survival or growth of L. alexandri. Cannibalism occurred during feed training. The worst survival, specific growth rate and high mortality were found with the use of extruded dry diet, while similar values were registered with the different feed training diets used. Reduced water flow rate can be used to lower water consumption during larviculture and feed training of L. alexandri.

Avaliaram-se os efeitos de diferentes fluxos de água e do condicionamento alimentar sobre a produção de juvenis de pacamã Lophiosilurus alexandri. Larvas com nove dias pós-eclosão (n=2.400) foram estocadas na densidade de 5 larvas/L. Diferentes fluxos (F) de água foram testados: F1 = 180; F2 = 600; F3 = 1.300; e F4 = 2.600mL/min. Náuplios de Artemia foram usados como alimento nos primeiros 15 dias de alimentação ativa. No segundo experimento, para o condicionamento alimentar, 720 juvenis (comprimento total de 22,2mm) foram estocados na densidade de 1,5 juvenis/L. Fluxo de água similar a F1 foi utilizado. Foram testados o uso de dieta extrusada e o condicionamento alimentar adicionado ou não de náuplios de Artemia ou emulsão Scott. Os diferentes fluxos de água não afetaram a sobrevivência e o crescimento de L. alexandri. Durante o condicionamento alimentar foi registrada a ocorrência de canibalismo. Pior sobrevivência, taxa de crescimento específica e maior mortalidade foram registradas para o uso da dieta comercial, enquanto valores similares foram verificados para as diferentes dietas usadas no condicionamento alimentar. Fluxo de água reduzido pode ser empregado durante a larvicultura e condicionamento alimentar de L. alexandri.