Common carp ( Cyprinus carpio L.) is one of the most intensively farmed fish species globally (FAO, 2020). In Europe, traditional multi-pond production lasts depending on temperature of water approx. 33 months and include two wintering stages. In the autumn of the first and second year fish are harvested and transferred to deep (2 - 2.5 m) earthen ponds until all maintenance works are completed in the production ponds, the ponds are inundated and the appropriate level of water temperature is reached, usually in April or May. One of the GAIN project (https://www.gain2020.com/ ) objectives included ecological intensification of C. carpio farming by reducing the time needed to produce market size fish (1 – 1.5 kg) from 33 to 19 months. This has been possible by transferring common carp fingerlings to a RAS (Recirculating Aquaculture System) for overwintering (October – May) and afterwards moving the fish to on-growing ponds in the autumn of the second production year . However, wintering of common carp in RAS under an ambient temperature is a new concept and it is of paramount importance to assess health and welfare indicators in complement to zootechnical indices . Our studies revealed that molecular and histological characteristics of the intestine assured successful assessment of nutritional status in common carp and shed light on the importance of gut microbiota composition and intestine brain interplay as wellbeing and health parameters (Eljasik et al., 2021). Therefore, the aim of the multifaced study was to assess intestine morphology, gut-brain axis (GBA) gene activity and composition of gut microbiota in C. carpio overwintering in RAS compared to earthen ponds.
Material and methods
The study was conducted during 2019/2020 wintering period (October – May) in RAS system (6 tanks, 3 m3 each, 100 fish per tank) under the tent and in earthen ponds (4.1 ha, density approx. 850 kg of carp per ha) of the Maliniec carp farm. Starting weight of fish in RAS and wintering ponds was approx. 45 ± 5 g. Fish in RAS were automatically fed two feed blends of different level of protein and fat, i.e., AL (42/12) and AG (30/9), while those in the ponds (ML) were not fed. Upon completion of the wintering AL, AG and ML fish (n = 5, each variant) were sacrificed, weighted and sampled, i.e. (i) proximate intestine to assess height of the mucosal folds (MS), width of lamina propria (LM), thickness of sub-epithelial mucosa (SM), number and area of goblet cells (GC), morphology of supranuclear vacuoles (SNV) and presence of eosinophilic v acuoles (EV); (ii) intestine and brain samples to assess health [e.g. occludin , claudin-3c , interleukin 6 (il6 ), mucin 5b (muc5b), 70 kDa-heat shock protein (hsp70 ) in the intestine] and GBA-related welfare [ e.g. γ-glutamyl transpeptidase (ggt ), cholecystokinin (cck ) in the intestine and agouti-related protein (agrp ), glucose transporter 2 (glut2 ), orexin in the brain] based on the expression of total 17 genes; and (iii) proximate intestine to characterise microbial communities by V3/V4 regions of the 16S r RNA gene sequencing with the Illumina MiSeq (300 PE), followed by raw data processing using DADA2 and analysis of composition of the gut microbiome using phyloseq in R package.
Results and Conclusions
Results from the histological evaluation showed that the AG fish had significantly ( P = 0.01) longer MF (1108.9 ± 48,33 µm), bigger SNV sizes (23.2 ± 3.13 µm), lower LP sizes (19.3 ± 3.93 µm), lower GC numbers (28.0 ± 2.45) and lower SM thickness (45.1 ± 8.31 µm) comparing to AL (1012.8 ± 50.7; 18.8 ± 1.67; 24.5 ± 5.16; 57.6 ± 3.44; and 59.1 ± 7.07) and ML (898.3 ± 64.09; no SNV; 42.4 ± 11.69; 73.5 ± 7.77; 68.8 ± 13.23). The GC area was significantly ( P = 0.01) larger among AG (50.7 ± 9.95 µm2) and ML (50.7 ± 13.54 µm2) fish comparing to AL (41.7 ± 10.63 µm2 ) fish. All together histopathological indicators showed that AG fish overwintering in RAS were in better condition compared to AL common carp, and also unquestionably to ML fish from the typical wintering pond. The main reason for the poor health condition of the ML fish from the Maliniec farm was ceased feeding that eventually induced enteritis characterized, among other assessed parameters, by the absence of SNV and presence of eosinophilic vacuoles in the LP . Intestinal morphology in AL fish confirmed that common carp fed diet with higher fat content (12%) comparing to AG (9%) had lower condition presumably due to excessive amount of energy in the feed in relation to the needs during overwintering. Resul ts of the gene expression analysis confirmed histological evaluation. Fish from the wintering ponds with clear symptoms of enteritis had the highest activity of occludin and claudin-3c both important in the assembly and maintenance of tight-junctions betwe en enterocytes. Moreover, downregulated expression of akp , ggt , muc5b and Na+/K+ ATPase in ML fish showed low metabolic activity due to lack of feeding during the wintering period. Conversely, activity of those genes was significantly higher in AL common carp fed on a high-fat and -protein diet. However, the AL fish had also elevated expressi on values of il-6 , il1b, hsp70 and claudin-3c genes all together involved in inflammation process. In case of AG fish, results showed intermediate activity of genes involved in regulation of nutrients metabolism and functioning of the physical intestinal b arrier, and low expression of pro-inflammatory genes. Our study, for the first time, characterised gut-brain axis in C. carpio . Activity of genes related to higher nutrient metabolism (orexin , agrp , glut2) had significantly higher expression in the brain of fishes from the AL group fed on a high-fat diet compared to fishes from the AG and ML groups . The analysis of microbiome based on V3/V4 regions revealed that composition of bacteria present in common carp gut was dominated by Proteobacteria and Firmicutes at the phylum level. Such microbial profile was previously described in literature for common carp (Liu et al., 2016), what may suggest no negative impact of wintering in RAS on common carp microbiota. Furthermore, on family level, microbiome was conquered by Entero bacteriaceae, particularly from genus Escherichia-Shigella and Enterococcus.
Concluding, wintering of common carp in RAS is a viable alternative to earthen ponds in which fish develop severe signs o f enteritis, and thus lower condition before grow-out phase. The multifaceted study also provided first characteristics of the interplay within the gut-brain axis in common carp, crucial to assure optimal level of health and welfare.
This work was supported by the Horizon 2020 fu nding under grant agreement No. 773330.
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