Co-Feeding of Pacu,Piaractus mesopotamicus Holmberg (1887), Larvae with Artemia Nauplii and a Microencapsulated Diet

Abstract

Aiming at a precocious substitution of live prey by artificial diet, a 20-day experiment with pacu, Piaractus mesopotamicuslarvae using co-feeding and abrupt weaning strategies was set up. At the end of the experiment, larvae fed Artemia showed the best results (P < 0.05) in weight, total length and biomass, compared with other treatments. Larvae fed exclusively a microencapsulated diet never ingested the diet. Diet ingestion in co-fed and abrupt-weaned larvae was low, but did increase during the experiment; however, Artemia influenced diet ingestion on co-fed larvae. Careful considerations should be given to diet processing and formulation to ensure survival and growth of larvae fed exclusively on prepared diets.     

Acute toxicity of ammonia in Pacu fish (Piaractus mesopotamicus,Holmberg, 1887) at different temperatures levels

Piaractus mesopotamicus juveniles (total length 12 ± 0.5 mm) were exposed to different concentrations of ammonia‐N (un‐ionized plus ionized ammonia as nitrogen), using the static renewal method at different temperature levels (15, 20 and 25°C) at pH 7. The 24, 48, 72, 96 h LC₅₀ values of ammonia‐N in P. mesopotamicus juveniles were 5.32, 4.19, 3.79 and 2.85 mg L^?1 at 15°C; 4.81, 3.97, 3.25 and 2.50 mg L^?1 at 20°C; and 4.16, 3.79, 2.58 and 1.97 mg L^?1 at 25°C respectively. The 24, 48, 72, 96 h LC₅₀ values of NH₃‐N (un‐ionized ammonia as nitrogen) were 0.018, 0.014, 0.013, 0.009 mg L^?1 at 15°C temperature; 0.023, 0.019, 0.016 and 0.012 mg L^?1 at 20°C; 0.029, 0.026, 0.018 and 0.014 mg L^?1 at 25°C. The temperature increase from 15 to 25°C caused an increase of ammonia‐N susceptibility by 21.80%, 9.55%, 31.92% and 30.87%, after 24, 48, 72 and 96 h exposure respectively. Furthermore, we found that exposure of fish to ammonia‐N caused an elevation in total haemoglobin and blood glucose with an increase of 2 mg L^?1 concentration. Ammonia levels tolerated, especially in different temperatures levels, have important implications for the management of aquaculture.     

Response of juvenile pacu (Piaractus mesopotamicus Holmberg, 1887) to balanced digestible protein

This 45‐day work aimed to determine the response of juvenile pacu (Piaractus mesopotamicus) to balanced digestible protein (BDP) and to use these responses to determine whether the optimum economic levels of BDP would differ depending on the form in which the fish is sold. Six isoenergetic diets containing 163, 201, 238, 272, 315 and 348 g/kg BDP (dry‐matter basis) were prepared through the serial dilutions of a high‐protein diet with the low‐protein diet. Fish (initial average body weight, 10.82 ± 0.14 g) were fed with respective experimental diets three times a day until apparent satiation. The optimum biological level of BDP was calculated as 326 g/kg (dry‐matter basis) by the quadratic regression model for maximum body weight gain of juvenile pacu. To maximize economic returns (US$/kg) for different end products, the optimum economic levels of BDP were calculated as 311, 317 and 319 g/kg (dry‐matter basis) by an economic model for whole‐body, eviscerated and sliced juvenile pacu respectively. This finding revealed that obtaining maximum biological performance of fish in the case of high feed costs or low prices of the end product in consumer market would substantially reduce the economic returns.     

Growth performance and energetic metabolism of pacu, Piaractus mesopotamicus (Holmberg, 1887), fed diets supplemented with ammonium metavanadate

Vanadium compounds mimic most of the metabolic effects of insulin, suggesting that it might be useful to improve utilization of dietary carbohydrate. This work evaluated the effect of dietary ammonium metavanadate (H₄NO₃V) on the growth performance and energy metabolism of pacu, an omnivorous South America characin. Two hundred and eighty‐eight fish were distributed into four blocks according to the body weight (21.8±1.7, 28.5±2.0, 28.4±1.9, 35.7±1.9 g), stocked in 24 plastic tanks and fed twice daily with isonitrogenous and isoenergetic diets containing six levels of H₄NO₃V (0, 10, 50, 100, 300 and 1000 mg kg^?1) for 60 days. Increasing levels of dietary ammonium metavanadate did not improve growth (P>0.05), and the highest level of inclusion (1000 mg kg^?1) reduced performance (P<0.05). Blood glucose levels decreased (P<0.05) in fish fed 300 and 1000 mg kg^?1 H₄NO₃V, but no differences were observed in other blood metabolites. A slight increase in muscle lipid content was observed in fish fed a diet containing 300 mg kg^?1 H₄NO₃V. Based on the results of this study, there is no benefit in supplementing pacu diets with metavanadate.     

Digestibility of energy,lipids and fatty acids of vegetable oils and poultry fat by pacu Piaractus mesopotamicus (Holmberg 1887)

Evaluation of vegetable oils and poultry fat digestibility is the first step to elicit their use in aquafeeds. This work aimed at determining apparent digestibility coefficients (ADCs) of energy, lipids and fatty acids of oil sources for pacu, a widely farmed neotropical Characin. A semipurified, omnivorous fish diet (344.2 g kg^?1 crude protein; 18.16 MJ kg^?1 gross energy) was used as reference diet. Test diets were obtained by adding 2 g kg^?1 chromium III oxide and replacing 15 g kg^?1 reference diet with fish, soybean, colza, corn and flaxseed oils and poultry fat. Juvenile pacu (64 ± 10.8 g; 14.6 ± 1.1 cm) were fed to apparent satiety, four times a day, and then transferred to cylindrical–conical aquaria for collection of faeces by sedimentation (n = 3). Apparent digestibility coefficients of energy and lipids were high for all tested oils (P > 0.05); ADCs of saturated fatty acid (SFA) were lower than monounsaturated fatty acids (MUFA) and polyunsaturated fatty acid (PUFA). Essential fatty acids (18:2n‐6 and 18:3n‐3) had high ADC (>93%), colza oil and poultry fat yielding the lowest ADC for 18:2n‐6 (P = 0.01) and 18:3n‐3 (P < 0.01), respectively. Corn oil, soybean oil and flaxseed oil were interesting sources of 18:2n‐6 and 18:3n‐3 dietary fatty acids for pacu.     

Free- and peptide-based dietary arginine supplementation for the South American fish pacu (Piaractus mesopotamicus)

Arginine was hypothesized to be a model compound in the present study on molecular forms of indispensable amino acid (IAA) dietary supplementation. Juvenile South American pacu (Piaractus mesopotamicus) were fed diets containing arginine in a protein base (casein‐wheat gluten or casein‐gelatin), or the casein‐wheat gluten base supplemented with dipeptide or free arginine at two levels (5 and 10 g kg^?1). Growth and protein efficiency ratios were significantly affected by diets, but not by arginine molecular form. Three free dispensable amino acids (DAA) and four IAA in plasma were affected by diet, but plasma arginine concentrations did not differ. Plasma urea concentrations, being very low in the pacu, and hepatic arginase activities, were not affected by diet (P = 0.10–0.11), but together with plasma ornithine, mirrored the growth data. Molecular form of arginine supplementation, free or dipeptide, significantly changed several free IAA (Phe, Leu, Ile, His) and urea, with a higher mean plasma concentration in dipeptide fed fish. The dietary treatments, or molecular form of the arginine supplementation, did not change proximate composition, except that calcium levels decreased with higher dietary arginine supplementation level. The present study indicates that dipeptides can provide IAA to pacu, and that arginine supplemented in this form is utilized as efficiently as in free form.     

Growth performance and body composition of pacu Piaractus mesopotamicus (Holmberg 1887) in response to dietary protein and energy levels

Improper dietary protein and energy levels and their ratio will lead to increased fish production cost. This work evaluated effects of dietary protein : energy ratio on growth and body composition of pacu, Piaractus mesopotamicus. Fingerling pacu (15.5 ± 0.4 g) were fed twice a day for 10 weeks until apparent satiation with diets containing 220, 260, 300, 340 or 380 g kg^?1 crude protein (CP) and 10.9, 11.7, 12.6, 13.4 or 14.2 MJ kg^?1 digestible energy (DE) in a totally randomized experimental design, 5 × 5 factorial scheme (n = 3). Weight gain, specific growth rate increased and feed conversion ratio (FCR) decreased significantly (P < 0.05) when CP increased from 220 to 271, 268 and 281 g kg^?1 respectively. Pacu was able to adjust feed consumption in a wide range of dietary DE concentration. Fish fed 260 CP diets showed best (P < 0.05) protein efficiency ratio and FCR with 11.7–12.6 MJ kg^?1; but for the 380 CP‐diets group, significant differences were observed only at 14.2 MJ kg^?1 dietary energy level, suggesting that pacu favours protein as energy source. DE was the chief influence on whole body chemical composition. Minimum dietary protein requirement of pacu is 270 g kg^?1, with an optimum CP : DE of 22.2 g MJ^?1.     

Cellular activity and development of enteric,hepatic and muscle tissues of juvenile pacu Piaractus mesopotamicus (Holmberg 1887) fed with lyophilized bovine colostrum

Bovine colostrum, an alternative food of animal origin provided as partial protein source, may constitute an innovative ingredient in fish feeding. Serum insulin‐like growth factor 1 (IGF‐1) and cellular activity of enteric, hepatic and muscle tissues of juvenile pacu Piaractus mesopotamicus fed with lyophilized bovine colostrum (LBC) were evaluated. Pacu (8.46 ± 0.74 g; 7.75 ± 0.27 cm, n = 18 in triplicate) were stocked into 18 tanks (300 L; 18 fish per tank, in triplicate) and fed with control diet containing 0% of LBC or experimental diet containing 10% and 20% of LBC inclusion for either 30 or 60 days of experiment. IGF‐1 serum concentration was not affected (P > 0.05) by the diet containing LBC and experimental period. The intestinal and hepatic cellular activity was not influenced by LBC (P > 0.05); however, intestine protein/RNA decreased at 60 days (P < 0.05). The muscle cellular activity was affected by LBC, highest in fish fed with diet containing 10% of LBC (P < 0.05). Between the periods DNA and protein/RNA increased at 60 days, while RNA, protein/DNA and RNA/DNA ratio decreased in this same period (P < 0.05). Increased DNA muscle content indicates fish growth by hyperplasia. The results of the present study is related with suitable development of enteric, hepatic and muscle tissues and proper condition of juvenile pacu fed with LBC considering the absence of differences in relation to juveniles that consumed diet without colostrum.     

Growth Rate of Pacu,Piaractus mesopotamicus,Fingerlings Fed Diets Containing Co-Dried Fish Silage as Replacement of Fish Meal

Abstract

Fish meal has been the most common animal protein source used to feed fish, even though it is fairly expensive and seasonally available. The objective of this work was to investigate co-dried fish silage as an alternative animal protein source for diets of pacu, Piaractus mesopotamicus.The diets did not differ significantly (P >0.05) with respect to average weight gain, ration consumption, apparent feed conversion, protein efficiency and specific growth rates. However, apparent protein digestibility and protein productive values were significantly different (P <0.01). Diets containing fish silage, co-dried with agricultural by-products (soybean meal or broken rice), showed better results than the control diet, which contained fish meal as the only animal protein source. The results showed that alternative ingredients can be used effectively to replace fish meal in pacu diets. Co-dried silages are economically and environmentally advantageous, since they use residues generated in the farm.     

Bovine plasma protein as an alternative protein source in extruded aquafeeds for Piaractus mesopotamicus (pacú) at two fattening stages

An 84‐day feeding trial was conducted to evaluate the addition of bovine plasma protein concentrate (BPP) as a full replacement of fish meal (FM) in extruded aquafeeds for the fish Piaractus mesopotamicus at two fattening stages. Fish with an initial body weight of 105.55 ± 13.48 g (1st fattening stage) and 564.50 ± 88.02 g (2nd fattening stage) received two iso‐nitrogenous and isocaloric diets with 130 g/kg of FM (FM diet—FMD) or with 130 g/kg of BPP (BPP diet—BPPD). No significant difference in growth performance was detected between dietary treatments. Fish from the 2nd fattening stage showed decreased intestinal pH and increased haemoglobin content. At the 1st fattening stage, fish fed with BPPD presented higher levels of plasma triglycerides and cholesterol, and hepatic and whole‐body lipid content, while plasma glucose and muscular glycogen were lower at the 2nd fattening stage compared with FMD‐fed fish. Fish fed with FMD showed an increase in the thiobarbituric acid reactive substances in liver at the 1st fattening stage and intestine at the 2nd one, accompanied by higher levels of hepatic catalase at the 1st fattening stage. Results suggest BPP could completely replace FM in diets for P. mesopotamicus without affecting growth performance, when FM represents 13% of the diet.     

Depletion study and estimation of withdrawal periods for florfenicol and florfenicol amine in pacu (Piaractus mesopotamicus)

The intensive production of farmed fish is at a global all‐time high, and the control of bacteria proliferation in fish farms requires the frequent use of antimicrobials. This practice raises important environmental concerns related to the emergence of antimicrobial‐resistant bacteria strains. Only a few antimicrobial drugs have been approved for use in aquaculture, one of which is florfenicol. This work studies the depletion and withdrawal period of florfenicol and its main metabolite, florfenicol amine, in pacu (Piaractus mesopotamicus), a neotropical characin widely farmed in the southern hemisphere. Juvenile pacu (average weight of 724 g) were stocked in a closed‐loop laboratory system with controlled water temperature (25.8°C), and were fed for 10 consecutive days with a diet containing an intended dose of 10 mg/florfenicol per kg bw. Muscle and skin tissues were collected at 1, 3, 6, 8, 10, 12 and 16 days post‐treatment, and florfenicol and florfenicol amine were quantified using a validated ultra‐high performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) method. The limits of quantitation for florfenicol and florfenicol amine were 10 ng/g in muscle and 50 ng/g in skin. Considering a maximum residue limit of 1000 ng/g for the sum of florfenicol and florfenicol amine in muscle with skin in natural proportions a withdrawal period of 5 days (water temperature 25.8°C) or 129 degree days was calculated on the basis of the upper limit of the one‐sided 95% confidence interval for the 99^th percentile derived from the residue depletion study.     

The effect of dietary protein level on pond water quality and feed utilization efficiency of pacúPiaractus mesopotamicus (Holmberg, 1887)

The effect of supplemental feeds with different levels of crude protein on pond water quality and food utilization efficiency by pacú (Piaractus mesopotamicus) was evaluated in a semi‐intensive culture system. Fish were stocked at a density of one individual per m², raised for 299 days, and fed isocaloric diets containing soybean meal, blood and bone meal, and yellow maize. Dietary treatments consisted of diets containing 25%, 35%, and 45% crude protein. Ponds receiving the two highest protein levels showed significantly higher values of alkalinity, conductivity and nitrites. High dietary protein diets resulted in no significant improvements in final weight (336.4±77.2, 308.2±92.6 and 368.4±82.2 g, mean±standard deviation, for 25%, 35% and 45% protein levels respectively). Moreover, fish carcass composition was not significantly different among treatments. Instead, significant augmentations in feed conversion ratio (1.36, 1.54 and 1.73 respectively) and reductions in protein retention (35.7%, 33.5% and 29.0% respectively) occurred as protein levels increased. Results indicate that at the lowest protein level, pacú growth rates and carcass composition were similar to the other experimental treatments, with a significant improvement in pond water quality and feed utilization efficiency.     

Growth and metabolism of pacu (Piaractus mesopotamicus Holmberg 1887) juveniles fed diets containing different protein, lipid and carbohydrate levels

To verify the potential of lipids and carbohydrates to spare dietary protein and to understand the intermediary metabolism of interaction of these nutrients in pacu juveniles, an experiment was carried out to evaluate pacu physiological and performance parameters. The experimental design was completely randomized with 12 treatments in a 2 × 2 × 3 factorial arrangement, consisting of diets containing two digestible protein levels (200 and 230 g kg⁻¹ PD), two lipid levels (40 and 80 g kg⁻¹) and three carbohydrate levels (410, 460 and 500 g kg⁻¹). Fish‐fed 230 g kg⁻¹ digestable protein (DP) showed increased glycaemia, decreased hepatic glycogen, as well as a smaller intake index and better feed conversion ratio. The higher dietary lipid level (80 g kg⁻¹) reduced protein intake and serum protein concentration, increased liver and body fat content, but did not affect growth. At a lipid level of 80 g kg⁻¹, the increase in dietary carbohydrate levels promoted greater weight gain (WG), crude protein intake (CPI) and better feed conversion ratio (FCR). For fish fed diets containing 40 g kg⁻¹ lipid, the best energy‐productive values (EPV) were obtained at 460 g kg⁻¹ carbohydrate. Increased levels of the main nutrients in the diets reduced the levels of serum triglycerides, while the increase in energy concentration increased the hepatosomatic (HSI) and glycaemia index values. Pacu used lipids as effectively as carbohydrates in the maximization of protein usage, as long as dietary protein was at a level of 230 g kg⁻¹ DP. The physiological parameters indicated that the best balance between the DP, dietary lipid and carbohydrate levels within the ranged this trial was obtained at 230, 40 and 460 g kg⁻¹, respectively, without lower growth.     

Kinetics of Cellular Component in Inflammatory Response Induced by Different Stimuli in the Swim Bladder of Pacu, Piaractus mesopotamicus Holmberg 1887 (Characidae)

Abstract.— The present study assessed the kinetics of cell accumulation at the site of inflammation induced by thioglycolate, Escherichia coli lipopolysaccharide (LPS) and heat-inactivated Aeromonas hydrophila , in the pacu, Piaractus mesopotamicus (Characidae), swim bladder. A quantitative, as well as qualitative, assessment was done of all the cells present in the exudate at 6, 24, and 48 h ( n  = 8) after inoculation of inflammatory agents. The results show that the thioglycolate was the irritant to induce higher total inflammatory cell accumulation when compared to the control group, 6 h after insult ( P  < 0.05). Inoculation of heat-inactivated Aeromonas hydrophila induced progressive accumulation of total inflammatory cells, with cell number peaking after 24 h and being significantly higher than observed in the other groups ( P  < 0.05). Injection of LPS also induced greater cell accumulation when compared to the control group ( P  < 0.05), although in lower numbers than those induced by the other two irritants. All irritants injected induced significantly greater accumulation of lymphocytes and thrombocytes when compared to the control group ( P  < 0.05).     

Growth performance of juvenile pacu (Piaractus mesopotamicus) and dourado (Salminus brasiliensis) fed with lyophilized bovine colostrum

Growth performance was evaluated in juvenile pacu (Piaractus mesopotamicus) and dourado (Salminus brasiliensis) fed diets containing 0%, 10% and 20% of lyophilized bovine colostrum for 60 days. The performance variables of weight gain, relative weight gain, feed intake, feed conversion ratio, specific growth rate, total protein intake and protein efficiency ratio were evaluated as well as apparent digestibility coefficients of protein and energy in the diets. Inclusion of bovine colostrum did not induce differences in the performance of pacu and dourado (P > 0.05), indicating that the diets were adequate and met the nutritional needs of the juveniles. The protein digestibility in both species was improved with the inclusion of the bovine lacteal secretion. Based on the variables studied, the diets with lyophilized bovine colostrum inclusion were nutritionally suitable for both pacu and dourado indicating the possibility to use this lacteal secretion as a partial substitute of dietary protein.     

Growth,nitrogen gain and indispensable amino acid retention of pacu (Piaractus mesopotamicus,Holmberg 1887) fed different brewers yeast (Saccharomyces cerevisiae) levels

A feeding‐and‐digestibility trials were carried out to evaluate the efficacy of replacing fishmeal with brewers yeast Saccharomyces cerevisiae in diets of pacu, Piaractus mesopotamicus, juveniles. The feeding trial was conducted during 54 days with 450 fish (26.6 ± 1.7 g) testing six isonitrogenous (270 g kg^?1 crude protein) and isoenergetic (19 MJ kg^?1 crude energy) diets, with increasing yeast level to replace 0 (control), 30, 35, 50, 70 or 100% of dietary fishmeal. Growth performance and feed utilization increased with increasing dietary yeast level until 50% fishmeal replacement. Protein retention efficiency was higher in fish fed 35 and 50%. Protein digestibility and the fillet hue (the red/green chromaticity) were not significantly different among all treatments. Nitrogen gains were significantly improved in fish fed 35% replacement diet compared to fish fed the control diet. The retentions of indispensable amino acids tended to increase with increasing dietary yeast levels, with maximum retention at 35–50%. On the basis of our results, replacing 50% fishmeal by yeast in pacu diets successfully improved feed efficiency and growth performance, and reduced nitrogen losses, thereby reducing the nitrogen outputs from fish farms.     

Stable carbon (δ13C) and nitrogen (δ15N) isotopes as natural indicators of live and dry food in Piaractus mesopotamicus (Holmberg, 1887) larval tissue

This study proposed the use of the stable isotope technique to track the type of food utilized by pacu Piaractus mesopotamicus larvae during their development, and to identify the moment when the larvae start using nutrients from the dry diet by retaining its carbon and nitrogen atoms in their body tissues. Five‐day‐old pacu larvae at the onset of exogenous feeding were fed Artemia nauplii or formulated diet exclusively; nauplii+formulated diet during the entire period; or were weaned from nauplii to a dry diet after 3, 6 or 12 days after the first feeding. δ¹³C and δ¹⁵N values for Artemia nauplii were ?15.1‰ and 4.7‰, respectively, and ?25.0‰ and 7.4‰ for the dry diet. The initial isotopic composition of the larval tissue was ?20.2‰ and 9.5‰ for δ¹³C and δ¹⁵N respectively. Later, at the end of a 42‐day feeding period, larvae fed Artemia nauplii alone reached values of ?12.7‰ and 7.0‰ for δ¹³C and δ¹⁵N respectively. Larvae that received the formulated diet alone showed values of ?22.7‰ for δ¹³C and 9.6‰ for δ¹⁵N. The stable isotope technique was precise, and the time at which the larvae utilized Artemia nauplii, and later dry diet as a food source could be clearly defined.     

Essential oils to control ichthyophthiriasis in pacu,Piaractus mesopotamicus (Holmberg): special emphasis on treatment with Melaleuca alternifolia

In vitro effect of the Melaleuca alternifolia, Lavandula angustifolia and Mentha piperita essential oils (EOs) against Ichthyophthirius multifiliis and in vivo effect of M. alternifolia for treating ichthyophthiriasis in one of the most important South American fish, Piaractus mesopotamicus (Holmberg), were evaluated. The in vitro test consisted of three EOs, each at concentrations of 57 μL L^?1, 114 μL L ^?1, 227 μL L^?1 and 455 μL L ^?1, which were assessed once an hour for 4 h in microtitre plates (96 wells). The in vitro results demonstrated that all tested EOs showed a cytotoxic effect against I. multifiliis compared to control groups (P < 0.05). The in vivo treatment for white spot disease was performed in a bath for 2 h day^?1 for 5 days using the M. alternifolia EO (50 μL L ^?1). In this study, 53.33% of the fish severely infected by I. multifiliis survived after the treatment with M. alternifolia (50 μL L ^?1) and the parasitological analysis has shown an efficacy of nearly 100% in the skin and gills, while all the fish in the control group died. Furthermore, the potential positive effect of M. alternifolia EO against two emergent opportunistic bacteria in South America Edwardsiella tarda and Citrobacter freundii was discussed.     

Whole-body amino acid pattern of juvenile,preadult, and adult pacu,Piaractus mesopotamicus,with an estimation of its dietary essential amino acid requirements

In the present study, juvenile (live body weight, 54.3 ± 8.2 g), preadult (live body weight, 822.5 ± 33.9 g), and adult (live body weight, 1,562.8 ± 41.8 g) pacu, Piaractus mesopotamicus, were used to estimate their dietary essential amino acid (EAA) requirements using the whole-body amino acid (AA) pattern. The results showed that whole-body moisture, crude protein, total lipid, and ash contents expressed on a wet weight basis (%) were significantly different among the studied growth phases. No significant differences were observed in the dietary EAA requirements estimated for the three growth phases of pacu. These dietary EAA requirements were found to be different than those previously estimated for the same fish through its muscle AA pattern. Based on whole-body EAA to total EAA ratios {A/E ratios; [(each EAA/total EAA) × 1,000]}, EAA requirements were estimated to be histidine (0.42%), arginine (1.36%), threonine (0.82%), valine (0.90%), methionine (0.45%), isoleucine (0.83%), leucine (1.29%), phenylalanine (0.74%), lysine (1.64%), and tryptophan (0.14%) for pacu. These estimated requirements may serve as a reference line in the formulation of practical and experimental diets until dose–response-based optimum EAA requirements are available for pacu.