Introduction
Landlocked Central Europe suffers from extremely low dietary omega-3 long chain polyunsaturated fatty acid (n-3 LC-PUFA) intake, specifically EPA+DHA. Pond-farmed carp (Cyprinus carpio) upcycle the limited EPA+DHA from the natural food web in freshwater ponds but can also endogenously fortify their concentrations under the temperate climate. The traditional grain-fed ponds produce carp fillets with low EPA+DHA, while feeding fish meal-fish oil at a high inclusion have profitability, sustainability, and water quality issues. Therefore, we aimed to validate the recently developed balanced pond feeding strategy for European fishponds to circumvent these problems, by using our pond fish nutrition supply–demand model as a guiding tool. The study had the following objectives: (1) a controlled in vivo nutritional bioenergetics trial comparing a formulated precision pond feed designed to maximize LC-PUFA retention against a wheat-fed control, and (2) a full-season pond trial using the precision pond feed under balanced pond feeding strategy as a proof-of-concept.
Materials and Methods
Laboratory trial: The "Pond feed" (PF) (31% protein, 9.80% lipid, 0.45% alpha-linolenic acid: C18:3n-3 or ALA, 2.61% linoleic acid: C18:2n-6 or LA, 5.33% saturated + monounsaturated fatty acid or SFA+MUFA, 33% non-starch polysaccharide or NSP, 20% Starch) was formulated via WinFeed using 70% low-opportunity-cost-biomass (corn DDGS, rapeseed and sunflower expeller meal, <5% freshwater fishmeal) and 30% food-feed conflict ingredients (whole wheat meal, rapeseed oil), meeting 90% of the ALA and LA requirement, with sufficient starch and lipids (1:1) as non-protein energy source to stimulate omega-3 sparing effect and higher LC-PUFA retention. This was compared against a traditional wheat control (WF) (9.5% protein, 1.80% lipid, 0.04% ALA, 0.69% LA 0.80% SFA+MUFA, 67.9% starch, 14.3% NSP) in a 70-day bioenergetics trial using common carp (Cyprinus carpio; ~20 g initial weight) in a Guelph-RAS system (8 tanks, 23–27 fish/tank; n=4/treatment), fed at 4% body weight/day in two rations. Uneaten feed and feces were collected to quantify actual consumption and whole-body fatty acid mass balance (retention, -oxidation, desaturation/elongation), via initial/final carcass and feed lipid/FA analysis. The trial ended at 70 days, once one treatment tripled its initial body weight. Whole-body fatty acid balance method was applied to understand the turnover of fatty acids in-vivo.
Pond trial: a pond validation trial with 2+ age carp (640–660 g) was conducted in 6 ponds at higher stocking density (625 kg ha���1; 975 individuals ha���1). Control ponds (C) (n=3) received wheat May–October; treatment (T) ponds (n=3) received wheat April–June and pond feed (PF) July–October, at equal total dose (~4252 kg���1 ha���1). Water quality and zooplankton biomass (200–500µm) were monitored monthly; harvested fish were analyzed for slaughtering indices, fillet lipid, and FA content. Statistical evaluation was pseudo-replication corrected accounting for the pond identity evaluating a mixed effect of supplementary feeding × pond itself.
Results
Laboratory trial: Both the n-3 and n-6 LC-PUFA biosynthesis rates were significantly higher in the PF than the WF group. In the n-3 LC-PUFA synthesis pathway, ��5/��6 desaturation (PF: 165.6±21.3 > WF: 48.6±39.2 nmol day-1 fish-1), elovl5 elongation (PF: 71.4±7.84 > WF: 23.5±18.5 nmol day-1 fish and elovl2 elongation (PF: 68.6±16.0 > WF: 10.9±11.0 nmol day-1 fish-1) were significantly (p<0.05) elevated in PF vs. WF. While both groups synthesized comparable EPA, PF synthesized significantly more DHA than WF (PF: 28.54–38.09 > WF: ���4.94–11.17 nmol DHA fish���1 day���1). Concurrently, in the n-6 LC-PUFA synthesis pathway, ��5/��6 desaturation (PF: 173.0±32.8 > WF: 29.3±2.63 nmol day-1 fish-1), elovl5 elongation (PF: 85.7±21.9 > WF: 17.5±2.44 nmol day-1 fish-1), was significantly (p<0.05) elevated in PF vs. WF; elovl2 activity was not observed in either group. However, enzymatic activities (elovl5, ��5/��6) toward n-3 and n-6 substrates did not differ significantly within either diet (p>0.05). Carp β-oxidized SFA and MUFA for energy, at significantly (p<0.05) higher rates in PF vs. WF (SFA; PF: 23.5±3.24 > WF:3.20±0.20, MUFA; PF: 51.5±2.20 > WF: 9.81±0.62 nmol day-1 fish-1).
Pond trial:
Gill-filterable plankton biomass did not differ between pond groups during early season (4.30–4.43 mg DM l���1). However, it diverged significantly during end season (T: 4.97±5.14 >C: 2.24 ±2.84 mg DM l���1) with the commencement of the application of the precision pond feed in treatment. The whole season plankton consortia FA is under analyses. The final body weight of carp, fillet yield differed between control and treatment ponds, while survivability was the same (>95%). Fillet lipid content did not differ significantly between control and treatment (10.8 ± 0.67 to 10.89 ± 1.52 g 100 g���1 fillet). Selectively, n-3 LC-PUFA concentrations in fillet were significantly higher in treatment than the control (p<0.05), but n-6 LC-PUFA unchanged (p>0.05). The %RDI of EPA+DHA (recommended minimum intake: 250 mg EPA+DHA person���1 day���1) per 100 g portion size of carp fillets (with skin) was 56.59 ± 4.57% for control and 83.80±4.86% for treatment (p<0.01). The yield of EPA+DHA from treatment ponds was 1.607 ± 0.099 kg ha���1, significantly (p<0.01) higher than the control (0.743 ± 0.015 kg ha���1), equivalent to ~4060 and ~1883 fish oil capsules ha���1 year���1, respectively.
Discussion
The carp fed pond feed demonstrated an n-3 LC-PUFA sparing effect by the whole-body fatty acid balance method as well as higher retentions of EPA and DHA than the wheat-fed control group in laboratory settings. In ponds, the in-vivo mechanism was further complemented by presence of a higher natural-food in balanced fed ponds receiving the precision pond feed pellets instead of wheat. Thus, showing a synergistic effect during the end-of-season brought about by the pond feed (i.e., favoring efficient LC-PUFA bioenergetics in carp body) and the boosted natural food web (i.e., favoring enhanced LC-PUFA supply).
Acknowledgement
The study was funded by the Ministry of Education, Youth and Sports of the Czech Republic, OP JAK – Aquaculture for Future project (CZ.02.01.01/00/23_021/0012616).