Slaughter is considered an important fish welfare issue. For a slaughter method to be considered humane, effective stunning that lasts until death as well as the reduction of pain and fear throughout all procedures are essential. Our objective was to investigate current techniques for slaughtering fish in Brazil. A digital questionnaire with six multiple-choice and three open questions was distributed to companies registered as fish slaughterhouses and to fish farms in 2019, addressing fish species, quantities processed and slaughter procedures adopted. From 62 facilities in 15 Brazilian states that answered the survey, nine slaughterhouses and 30 fish farms slaughtered fish, totalling 452 tonnes per day. All reported pre-slaughter stunning, and live chilling to be the most commonly cited method (82.0%), followed by electronarcosis (18.0%). Slaughtering techniques included exsanguination (38.5%) and decapitation (2.5%). For the remaining companies, no slaughter method was declared, suggesting death by asphyxiation or by further processing (59.0%). Twenty-nine companies adopted pre-slaughter fasting, lasting from 10 to 48 h. All sites worked with tilapia (100%) and 24 (82.7%) reported that they worked exclusively with this species. Other species reported were: pacu (25.6%), tambacu (17.9%), tambaqui and carp (15.3%), jundiá (12.8%), pintado and pirarucu (7.7%), matrinxã and pangassius (5.1%). Asphyxia and live chilling are not considered humane, as animals remain conscious and, thus, suffer. Results show that most establishments do not perform humane slaughter. This scenario highlights the urgent need for development and enforcement of humane fish slaughter techniques, with routine supervision and normative requirements.

Poor utilisation efficiency of carbohydrate always leads to metabolic phenotypes in fish. The intestinal microbiota plays an important role in carbohydrate degradation. Whether the intestinal bacteria could alleviate high-carbohydrate diet (HCD)-induced metabolic phenotypes in fish remains unknown. Here, a strain affiliated to Bacillus amyloliquefaciens was isolated from the intestine of Nile tilapia. A basal diet (CON), HCD or HCD supplemented with B. amy SS1 (HCB) was used to feed fish for 10 weeks. The beneficial effects of B. amy SS1 on weight gain and protein accumulation were observed. Fasting glucose and lipid deposition were decreased in the HCB group compared with the HCD group. High-throughput sequencing showed that the abundance of acetate-producing bacteria was increased in the HCB group relative to the HCD group. Gas chromatographic analysis indicated that the concentration of intestinal acetate was increased dramatically in the HCB group compared with that in the HCD group. Glucagon-like peptide-1 was also increased in the intestine and serum of the HCB group. Thus, fish were fed with HCD, HCD supplemented with sodium acetate at 900 mg/kg (HLA), 1800 mg/kg (HMA) or 3600 mg/kg (HHA) diet for 8 weeks, and the HMA and HHA groups mirrored the effects of B. amy SS1. This study revealed that B. amy SS1 could alleviate the metabolic phenotypes caused by HCD by enriching acetate-producing bacteria in fish intestines. Regulating the intestinal microbiota and their metabolites might represent a powerful strategy for fish nutrition modulation and health maintenance in future.

Commercial diets for tilapia juveniles contain high levels of plant protein sources. Soybean meal has been utilised due to its high protein content; however, soy-based diets are limited in methionine (Met) and require its supplementation to fulfil fish requirements. dl-Methinone (dl-Met) and Ca bis-methionine hydroxyl analogue (MHA-Ca) are synthetic Met sources supplemented in aquafeeds, which may differ in biological efficiency due to structural differences. The present study evaluated the effect of both methionine sources on metabolism and growth of Nile tilapia. A growth trial was performed using three isonitrogenous and isoenergetic diets, containing plant ingredients as protein sources: DLM and MHA diets were supplemented on equimolar levels of Met, while REF diet was not supplemented. Hepatic free Met and one-carbon metabolites were determined in fish fed for 57 d. Metabolism of dl-Met and MHA was analysed by an in vivo time-course trial using 14C-labelled tracers. Only dl-Met supplementation significantly increased final body weight and improved feed conversion and protein efficiency ratios compared with the REF diet. Our findings indicate that Met in DLM fed fish follows the transsulphuration pathway, while in fish fed MHA and REF diets it is remethylated. The in vivo trial revealed that 14C-dl-Met is absorbed faster and more retained than 14C-MHA, resulting in a greater availability of free Met in the tissues when fish is fed with DLM diet. Our study indicates that dietary dl-Met supplementation improves growth performance and N retention, and that Met absorption and utilisation are influenced by the dietary source in tilapia juveniles.

The present study aimed to investigate nutritional programming of carbohydrate metabolism in Nile tilapia. Early nutritional intervention stimulus was achieved by feeding fry with high-protein/low-carbohydrate (HP/LC) or low-protein/high-carbohydrate (LP/HC) diet since first feeding for 4 weeks, and the effect of nutritional stimulus on carbohydrate and its related metabolism was evaluated through the adult stage. Our findings indicated that at week 1, LP/HC diet-fed fry had lower levels of mRNA for genes coding gluconeogenesis and amino acid catabolism and higher levels of hk2 (P < 0⋅05). As expected, in adult tilapia, although LP/HC diet-fed fish had poorer growth (end of stimulus), the fish showed compensatory growth. There were permanent effects of early high-carbohydrate (HC) intake on several parameters, including (1) modulating hepatic composition, (2) increased muscle glycogen, (3) lower levels of enzymes involved in amino acid catabolism and (4) higher levels of glycolytic enzymes in glycolysis. Finally, HP/LC diet- and LP/HC diet-fed fish were challenged with different dietary carbohydrate levels. Irrespective of challenging diets, the early HC stimulus had significant effects on adult tilapia by (1) promoting utilisation of glucose, which had protein-sparing effects for better growth, (2) inducting lipogenesis and (3) decreasing amino acid catabolism. Taken together, for the first time, we demonstrated that early HC feeding was effective for positive nutritional programming of metabolism in Nile tilapia (an omnivorous fish). It led to the improvement of growth performance in adult fish associated with early feeding, which is linked to a better ability to use glucose, to induce lipogenesis, and to suppress amino acid catabolism.

The fenofibrate functions in mammals could be affected by many factors such as dietary nutrient levels and physiological status. However, this phenomenon has not been well studied in fish. The goal of our study was to investigate the effect of dietary protein contents on metabolic regulation of fenofibrate in Nile tilapia. An 8-week experiment was conducted to feed fish with four diets at two protein levels (28 and 38 %) with or without the supplementation of fenofibrate (200 mg/kg body weight per d). After the trial, the body morphometric parameters, plasma biochemical parameters and quantitative PCR data were examined. These results showed that fenofibrate significantly reduced the feeding intake and weight gain rate, increased the oxidative stress (increased plasma methane dicarboxylic aldehyde) and liver : body ratio (increased hepatosomatic index) in the low protein (LP)-fed fish. In contrast, fenofibrate exhibited a lipid-lowering (reduced hepatic lipid) effect and up-regulated the expressions of the genes related to lipid catabolism, transport and anabolic metabolism in the high protein (HP)-fed fish. The present study suggested that lipid-lowering effect of fenofibrate would be strengthened in the fish fed with the HP diet containing high energy, but in the fish fed with the LP diet containing low energy, the fenofibrate treatment would cause adverse effects for metabolism. Taking together, our study showed that the metabolic regulation of fenofibrate in Nile tilapia was dependent not only on feed energy content but also on dietary nutrient composition, such as dietary protein and/or lipid levels.

Probiotic administration can be a nutritional strategy to improve the immune response and growth performance of fish. The current study aimed to evaluate the effects of a probiotic blend (Bacillus sp., Pediococcus sp., Enterococcus sp., Lactobacillus sp.) as a dietary supplement on growth performance, feed utilization, innate immune and oxidative stress responses and intestinal morphology in juvenile Nile tilapia (Oreochromis niloticus). The probiotic was incorporated into a basal diet at three concentrations: 0 g/kg (A0: control), 3 g/kg (A1: 1.0×106 colony forming unit (CFU)/g) and 6 g/kg (A2: 2.3×106 CFU/g diet). After 8 weeks of probiotic feeding, weight and specific growth rate where significantly higher in fish-fed A1 diet than in fish-fed A0. Alternative complement in plasma was significantly enhanced in fish-fed A2 when compared with A0. The hepatic antioxidant indicators were not affected by probiotic supplementation. Villi height and goblet cell counts increased significantly in the intestine of fish-fed A1 and A2 diets compared with A0. The dietary probiotic supplementation was maintained until 20 weeks of feeding. Then the selected immune parameters, digestive enzymes and apparent digestibility of diets were studied. No effect of probiotic feeding was observed after that longer period supplementation. The dietary supplementation of mixed species probiotic may constitute a valuable nutritional approach towards a sustainable tilapia aquaculture. The improvement of the immune responses and intestinal morphology play an important role in increasing growth performance, nutrient absorption and disease resistance in fish, important outcomes in such a competitive and developing aquaculture sector.

The effect of the type of non-protein energy (NPE) on energy utilisation in Nile tilapia was studied, focusing on digestible energy utilisation for growth (kgDE). Furthermore, literature data on kgDE across fish species were analysed in order to evaluate the effect of dietary macronutrient composition. A total of twelve groups of fish were assigned in a 2 × 2 factorial design: two diets (‘fat’ v. ‘starch’) and two feeding levels (‘low’ v. ‘high’). In the ‘fat’-diet, 125 g fish oil and in the ‘starch’-diet 300 g maize starch were added to 875 g of an identical basal mixture. Fish were fed restrictively one of two ration levels (‘low’ or ‘high’) for estimating kgDE. Nutrient digestibility, N and energy balances were measured. For estimating kgDE, data of the present study were combined with previous data of Nile tilapia fed similar diets to satiation. The type of NPE affected kgDE (0·561 and 0·663 with the ‘starch’ and ‘fat’-diets, respectively; P < 0·001). Across fish species, literature values of kgDE range from 0·31 to 0·82. Variability in kgDE was related to dietary macronutrient composition, the trophic level of the fish species and the composition of growth (fat:protein gain ratio). The across-species comparison suggested that the relationships of kgDE with trophic level and with growth composition were predominantly induced by dietary macronutrient composition. Reported kgDE values increased linearly with increasing dietary fat content and decreasing dietary carbohydrate content. In contrast, kgDE related curvilinearly to dietary crude protein content. In conclusion, energy utilisation for growth is influenced by dietary macronutrient composition.

A 5′-flanking region and partial open reading frame (ORF) of the β-actin gene (GenBank accession No. EF026001) of Nile tilapia (Oreochromis niloticus) was cloned by polymerase chain reaction (PCR) amplification. The segment included a 1643 bp regulatory sequence and a 90 bp partial ORF which encoded a 30-amino-acid peptide. The regulatory sequence comprised a 108 bp 5′ proximal promoter, the first untranslated exon and the first intron of the β-actin gene. The proximal promoter region contained elements that were critical for transcription activity, including a CCAAT, TATA and CArG box located at –92, –29 and –62 bp upstream of the transcription initiation site, respectively. The regulatory sequence was inserted into the promoterless pDsRed2-1 vector to construct the expressing vector pNA-DsRed. The linearized pNA-DsRed was microinjected into the fertilized eggs of Tanichthys albonubes. The expression of the DsRed2 gene in transgenic fish could be observed under the microfluoroscope and anatomical lens. The results showed that the β-actin gene promoter possessed effective transcription activity.

The effects of nitrite (NO2–) on the Nile tilapia (Oreochromis niloticus) were investigated, due to their relevance to worldwide aquaculture. Hematological parameters – functional haemoglobin (oxy+deoxy-Hb, %), methaemoglobin (MetHb%), and hematocrit (Hct) – and – plasma osmolality, plasma chloride ([Cl−]) and magnesium ([Mg2+]) concentrations – were analysed. Two experiments were conducted, each with its own non-contaminated control, 8 tilapias for each group. In experiment 1 (48 hours), and the first 48 hours of experiment 2, fish were contaminated with 0.4 mM NO2– (added as NaNO2). In experiment 2, ambient NaNO2– was removed for the second 48 hours. NaNO2– exposure increased plasma [NO2–] to 0.4 mM, which increased MetHb from 10.8 ± 2.5% to 46.8 ± 8.0%, and consequently decreased functional Hb from 89.2 ± 2.5% to 53.2 ± 8.0%. NaNO2 removal led to recovery of both parameters. Both Hct and plasma Mg2+ were lowest in fish recovering from nitrite exposure. Thus, these parameters did not show recovery. Plasma osmolality and [Cl−] were not affected by NaNO2–, probably due to the moderate rise in plasma [NO2–]. O. niloticus was thus sensitive to 48 hours of exposure to 0.4 mM NaNO2, and partially recovered from its effects after 48 hours in sodium nitrite-free water. Cultivators of Nile tilapia should thus consider the toxicity of nitrite, especially in aquaculture systems using recirculating water.