Introduction
In most European fishponds culturing cyprinids, natural food and supplemented cereals such as wheat is the business as usual. The protein and indispensable amino acid (IAA) quantity x quality of natural food webs fluctuate seasonally. The carp growth, N:P retention or excretion decisions are at the mercy of that, ranging from very good to very bad across the vegetative season. The supply-demand dynamics of some critically limiting IAAs such as lysine (LYS) and methionine (MET) remain poorly understood, as part of nutritional ecology of aquaculture ponds. It is not only the case of European fishponds, but also a globally relevant question. The present study had two objectives. Pre-trial: to develop a model of supply-demand of LYS and MET under traditionally grain-fed (<10% CP) or other low-protein pellet-fed ponds (<25% CP). Main-trial: to develop and test a precision-balanced pond feed (at the threshold of protein feed nomenclature, i.e., 30% CP) that solves the supply-demand gap of LYS-MET in ponds through a carp nutritional bioenergetics assay and simultaneous validation in pond for proof-of-concept.
Materials and Methods
Pre-trial: Two groups of ponds were selected (Group-A, n=3 ponds: fed by wheat alone, 10% CP during April-October; Group-B, n=6 ponds: fed by wheat during April–June and low-protein plant-pellet 23% CP during July–October, either by hand or demand-feeder) at the Faculty of Fisheries and Protection of Waters research station, Vodnany stocked with stocked with 2+ age Cyprinus carpio (384.82±3.47 g; 471 kg ha���1; 1183±23 individuals ha-1). Monthly plankton consortia samples were collected from nine earthen experimental ponds (0.16 ha area, 0.8 m depth), using a 200 µm-mesh plankton net (mimicking branchial sieve minima of common carp >200 g), lyophilized, and stored at ���20 °C. Samples were hydrolyzed using two procedures: acid hydrolysis (6 M HCl, 108 °C, 23 h) for total AAs, and oxidative hydrolysis (formic acid/H2O2 followed by 6 M HCl, 120 °C, 23 h) for cysteine and methionine. After evaporation and reconstitution, AAs were separated by ion-exchange chromatography on an AAA 500 analyzer with post-column ninhydrin derivatization and photometric detection at 570 nm and 440 nm.
Main trial: precision pond feed (31% CP) was formulated in WinFeed using 70% LOCB (low-opportunity-cost-biomass, comprising corn DDGS, rapeseed expeller meal, sunflower expeller meal, and <5% freshwater fishmeal from fish processing) and 30% human feed-food conflict ingredients (such as wholewheat meal and rapeseed oil). The IAA profile was carefully calibrated using crystalline L-lysine hydrochloride and DL-methionine (maintaining stoichiometry 3:1) in a way that the IAA profile mimics zooplankton-zoobenthos standards (in terms of DIAASLYS,MET >50%) and the feed fulfills ≥90% of LYS, MET requirements of carp. The starch (21%) and lipid (8%) were carefully balanced to fulfill ≥100% non-protein energy requirement of carp. It also has enough starch (>20%) for high-quality extrusion of pellets. A high non-starch polysaccharide (NSP 33%) contributed by LOCBs were accepted as filler but given that NDF:ADF ratio is >2 (for making the fibers easily fermentable in carp gut or by pond).
Precision pond feed was evaluated against wheat (as a control) for apparent digestibility, metabolic losses, and retention of protein in our Guelph-RAS system using standard methodologies. For this purpose, the lab trial lasted 70-day with common carp (initial body weight ~20g; 23–27 fish per tank) randomly assigned to 8 tanks across two treatments: pond feed and wheat with four replicates each (fed 6% BW day���1, split into two doses at 08:00 and 14:00) and uneaten pellets dried, measured (40 mins post feeding). Following clearing of uneaten pellets after 2nd feeding, passive faeces collection continued every 4 hours until next day feeding. Initial and final carcass were taken for whole-body homogenates for protein, making sure that the duration of experiment has passed until one of the treatments had tripled body weight. Simultaneously, a pond validation trial with 2+ age carp (640–660 g) was conducted in 9 ponds (same as pre-trial) but challenged by a higher stocking density (625 kg ha���1; 975 individuals ha���1). Control ponds (n=3) received wheat during May–October. Same total dose treatment ponds (n=6) received as wholewheat or extruded breakfast cereals during April-June, and precision pond feed during July–October. Ponds were equipped with underwater temperature data loggers to precisely compute degree-days of culture, and fish measurements done monthly (n=20 pond���1 month���1) to compute inter-month thermal growth coefficient (TGC). Monthly monitoring of water quality (nutrients, chlorophyll-a) and zooplankton biomass (200–500 µm; >500 µm) was monitored.
Results
Pre-trial: During May–June, the supply of LYS (70–121 g) and MET (24–46 g) significantly exceeded (p<0.05) pond carp's demand (LYS: 32–59 g; MET: 9–18 g). July acts like a transition period when supply and demand of LYS and MET are in equilibrium (p>0.05). From August onwards, LYS supply fell to 34–54 g (p<0.05) against a demand of 91–115 g, and MET supply dropped to 15–24 g (p<0.05) against a demand of 27–34 g. This late-season drop coincided with decline of zooplankton (and zoobenthos) in pond, but an increase in algae (chlorophyll-a). Overall, the pattern shifted from early-season IAA surplus to late-season IAA limitation. Selecting any low protein level with any random IAA profile such as the low-protein pellet fed in group-B ponds (23% protein, fulfilling 40% of carp's LYS requirement, 66% MET requirement) over wheat fed in group-A ponds (10% protein, fulfilling 12% LYS requirement, 23% MET requirement) does not work. Such imprecise interventions do not solve the late-season IAA deficit of pond carp, as the net yield and relative feeding coefficient (RFC) of group-A and group-B ponds were same (p>0.05).
Main trial: Application of precision pond feed during the problematic period of IAA supply-demand (July–September) had solved the issue of depressed growth in carp, compared to control ponds. In control, the TGC of pond carp stock collapsed during July-September. In treatment, during the same period, the TGC of pond carp was significantly reinforced compared to control (p<0.05), that too with consistently high TGC across the months July to September (p>0.05). This led to significantly high net yield (1.7 tons ha���1), bigger market-sized carp (2.4 kg, market-size 2 kg already in September), and RFC (2.5) in treatment ponds (p<0.05) than the control ponds (1.2 tons ha���1; weight 1.9 kg; RFC 3.4). The water quality was also controlled. Despite such high production and application pellets in treatment pond, the end-of-culture (August–November) concentrations of nutrients (C: N:P) in water or sediment, and chlorophyll-a concentrations were same as control (p>0.05). The large-bodied zooplankton (>500 µm) biomass was significantly (p<0.05) enhanced in treatment (5–24 mg L���1) over control ponds (3–12 mg L���1) during August–October, indicating natural food could regenerate when deficiency for fish was lifted. A high bioenergetic efficiency of precision pond feed over wheat was confirmed in the lab trial. Protein storage efficiency of precision pond feed (net-stored protein 40% of digested protein) was significantly greater (p<0.05) than that of wheat (net-stored protein 30% of digested protein). Net-storage of amino acids from the digested pool in-vivo is presently under construction.
Acknowledgment: The study was funded by the following national grants of the Czech Republic: OP JAK CZ.02.01.01/00/23_021/0012616 and NAZV QK22010177.