Introduction

Common carp (Cyprinus carpio L., 1758) is one of the most important aquaculture species in the world and the most important in Central and Eastern Europe (Horváth et al. 2002; Roy et al. 2020). According to the FAO (2018), it was ranked third, with 4.56 million tons being produced yearly. Its aquaculture is spread worldwide in tropical, subtropical, and temperate regions. Carp is produced in intensive, semi-intensive, and extensive production systems. Semi-intensive systems are dominantly used for carp production in Serbia as in the rest of the world (Marković 2010). Compound feeds are not commonly used in common carp aquaculture (Gyalog et al. 2011), as they are more expensive than cereals. The reason is its financial advantage: fish are fed naturally present food, and additional feeds are supplemented according to fish needs and the presence of natural food. This allows higher yields than those in extensive systems at lower production costs compared to the more intensive production systems. Intensive production systems are characterised by increased profit per unit area but have a decreased profit margin (Naylor et al. 2000).

Common carp are omnivorous fish, and in fish-ponds they dominantly consume macrozoobenthos, zooplankton, detritus, and plant seeds, which are naturally available food (Sibbing 1988; Chapman and Fernando 1994; García-Berthou 2001). Additional feeds used in semi-intensive carp aquaculture are cereals and formulated diets. Formulated diets are mixtures of ground cereals enriched with oils, proteins, and vitamins of various origins (Marković et al. 2016). Some of the widely used ingredients in formulated diets are of plant-derived materials, such as legume seeds, different types of oilseed cakes, leaf meals, leaf protein concentrates, and root tuber meals, which contain a large spectrum of antinutritional factors (Francis et al. 2001). These factors together with toxins that can develop in feeds and ingredients due to mishandling, which are anti-quality factors, interfere with food utilisation and negatively affect the health and production of animals (Makkar 1993).

The functional microanatomy of the liver and intestine represent a valuable tool for the assessment of the effects of different feeds on fish nutritional physiology (Rašković et al. 2011). Common carp liver consists of several lobes that are brownish in colour and closely adhered to other organs in viscera, such as the spleen, intestine, and the bladder (Farag et al. 2014). The main building blocks of the liver are hepatocytes, which are polygonal in shape with centrally located nuclei and prominent nucleoli. Their size is dependent on the amount of stored nutrients (Roberts 2012; Caballero et al. 1999) and is often correlated with feed type (Poleksić et al. 2014b). The distal intestine of fish morphologically resembles a simple tube with a wrinkled inner surface. It has distinctive layers, starting from the inner to the outer diameter cross-section: mucosa - mucosal layer containing inner epithelium, lamina propria (a cellular connective tissue), and muscularis mucosae; submucosa - connective tissue; two-layered muscle coat; ending with serosa (Mumford et al. 2007; Genten 2009).

Red blood cell morphology is routinely used in fish physiological surveys, particularly in toxicology (Witeska 2013). Erythrocytes in fish are produced mainly in the spleen and head kidney (Homechaudhuri and Jha 2001). From these organs, immature erythrocytes are released into the bloodstream, where they mature to erythrocytes. Immature erythrocytes are irregularly round to oval, polychromatic, and smaller thanmature erythrocytes, which appear normochromatic (Kondera 2011). An abundance of immature erythrocytes is an indirect measure of erythropoiesis activity in fish (Rios et al. 2005). A reduction in the erythrocyte turnover rate may be a result of disease, malnutrition, or long periods of starvation (Witeska 2015).

This study was designed to follow-up on our previous studies (Rašković el al. 2015, 2016a, b) on the effects of additional diets in the semi-intensive culture system on the growth performance and digestive system histology of the common carp. Unlike previous studies, this experiment was performed in cages placed in a single pond. In addition to previous experiments, a control group of fish fed naturally available food exclusively was included; together with hepatocytes and intestinal fold structure, the morphology of erythrocytes was also evaluated.

Materials and methods

Experimental animals and feeds

Carps used in the experiment originated from the same batch, were one year old, and grown in an earthen pond system at the Centre for Fisheries and Applied Hydrobiology (CEFAH), University of Belgrade, Faculty of Agriculture. No ethical approval was required in order to conduct the trial.

The extruded diet (E) used in the experiment was a commercially available, extruded complete mixture for growing one-year-old juvenile carp and for the intensive growth of two-year-old juvenile carp with 38 % protein (SOPROFISH 38/12 INTENSIVE EFFECT, Veterinary Institute Subotica, Subotica, Serbia). A pelleted feed (P) was obtained from a commercial producer (DTD Ribarstvo, Bački Jarak, Serbia). Wheat (W) was obtained from a local grocery store, and no additional preparations were performed prior to giving it to the fish. Fish feeds’ chemical properties were analysed in the Institute of Chemistry, Technology and Metallurgy, Belgrade, Serbia, using accredited methods (Table 1).

Table 1 Chemical properties of used feeds
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Experimental design

The experiment was performed in a 0.216 m3 (0.6 × 0.6 × 0.6 m) square shaped cages covered with a 1 cm2 mesh net. The bottom of each cage was paved with a plastic sack to prevent food loss. A total of 12 cages were dipped in a 550 m2 earthen pond alongside hanging bridge across the pond (Fig. 1). The distance between the nearest cage was approximately 30 cm and between those on opposing sides was approximately 1 m.

Fig. 1
figure 1

Scheme of the experiment area. C, control group; W, wheat fed group; P, the group fed pelleted diet; E, group fed extruded diet; brown color, shore; blue color, water

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Preparation for the experiment included: (i) drying out the pond, disinfection with calcium carbonate (150 g m-2), and three days later, the pond was refilled with water, originating from ground wells at a 125 m depth; (ii) two weeks after the pond was filled, 33 fish, with a total mass of 3.5 kg, were added to the pond to induce normal carp pond turbidity conditions by stirring up the sediment. The experiment started one week after adding the fish.

Carp were randomly chosen and sedated with clove oil. Then, the length, weight, and height of each fish was measured. Fish were randomly allocated in groups and placed in cages. Groups were formed in triplicates, randomly designed with equal initial weight of the fish. In total, 15 fish (x̅ = 12.05 ± 0.09 g) were placed in each cage. Four different groups were made: (I) control group (C) fed exclusively on natural feed from the pond; (II) group W fed wheat; (III) group P fed a pelleted diet; (IV) group E fed the extruded diet. The experiment lasted for 80 days from 13th August to 30th October 2016.

Feeding took place every day at 9:00, when fish in each cage were fed 3 % of their total biomass. Feed was dropped above each cage where it slowly sank towards the bottom of a cage. Besides added diets, naturally present invertebrates were able to roam into cages through the mesh net. Immediately after feeding, temperature, pH, conductivity, oxygen concentration, and oxygen saturation were measured using a MULTI 340i/SET apparatus (WTW, Weilheim in Oberbayern, Germany) (Table 2).

Table 2 Mean values ± SD of physical and chemical parameters of water in the experimental pond
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The feeding rate was adjusted every 15 days, when body weight, body height, and total length of fish were determined. From these parameters, body weight gain (BWG), specific growth rate (SGR), and conditional factor (CF) were calculated using Eqs. (1), (2) and (3), respectively:

$$\mathrm{BWG}\ \left(\mathrm{g}\right)=\mathrm{final}\ \mathrm{body}\ \mathrm{mass}\ \left(\mathrm{g}\right)-\mathrm{initial}\ \mathrm{body}\ \mathrm{mass}\ \left(\mathrm{g}\right)$$
(1)
$$ \mathrm{SGR}\left(\%{\mathrm{day}}^{-1}\right)=\frac{\ln \left(\mathrm{final}\ \mathrm{body}\ \mathrm{mass}\ \mathrm{in}\ \mathrm{g}\right)\times \ln \left(\mathrm{initial}\ \mathrm{body}\ \mathrm{mass}\ \mathrm{in}\ \mathrm{g}\right)}{\mathrm{number}\ \mathrm{of}\ \mathrm{trial}\ \mathrm{days}}\times 100 $$
(2)
$$\text{CF} \left(\text{g cm}^{-3}\right)=\frac{\text{weight} \left(\text{g}\right)}{{\text{total length}}^{3} \left({\text{cm}}^{3}\right)}$$
(3)

Histological sample preparation and evaluation

Histological samples were collected at the end of the experiment after fish were anesthetised in water containing several drops of clove oil and sacrificed by cervical transection and pithing. Three fish were randomly selected from each cage, for a total of 9 per treatment.

Blood was sampled before the fish were sacrificed by puncturing the caudal vein using a syringe. Blood smears were air dried and stained with Romanowsky stain using a Bio-Diff Kit (BioGnost, Zagreb, Croatia). From each fish, two blood smears were made and stained. Subsequently, 10 microscopic view fields using 400× magnification from each slide were randomly selected using ImageJ software. On each field, three erythrocytes were systematically chosen by superimposing the counting frame at the centre of the image and measuring. The measured parameters were the long and short axes and surface area of each erythrocyte. Circularity was calculated using the following formula:

$$\text{Circularity}=4{\uppi } \frac{\text{surface area}}{{\text{perimeter}}^{2} }$$
(4)

Each fish was dissected, and a portion of the liver and distal intestine were sampled and fixed in 4 % formalin solution (Lach-Ner, Czech Republic). After fixation, samples were subsequently dehydrated in an ethanol series and treated with an xylene in automatic tissue processor (TP 1020, Leica, Nussloch, Germany). Samples were then embedded in paraffin wax and sectioned with a sliding microtome SM 2000R (Leica) to 5–7 μm thickness. Sections were fixed on glass slides and stained with haematoxylin and eosin (HE). From each slide, 10 systematically chosen micrographs were taken at random using a 400× magnification. Calculation of the relative volume density was performed using the combination of unbiased counting frames and point counting-intercept methods proposed by Gundersen et al. (1988). The number of points hitting each tissue layer [lamina propria, goblet cells, tunica mucosa, and intraepithelial macrophages (IEM)] was divided by the total number of points present in the grid net (212 in total).

For every intestine cross-section slide, a series of micrographs were taken at 100× magnification. Micrographs were merged into a single cross-section using the MosaicJ plug-in in ImageJ software. The intestinal folds, intestinal muscle layer thickness, and whole cross-section diameter were observed (Fig. 2). Cross-section diameter values are represented by the mean length of n = 10 cross-sections for each intestine sample.

Fig. 2
figure 2

a Erythrocytes from common carp blood smear; b hepatocytes from liver cross-section; c intestinal folds from intestine cross-section; d whole intestine cross-section. LA, longer axis; SA, shorter axis; LD, nucleus distance from the nearest cell membrane; M, intraepithelial macrophage; G, goblet cell; LE, lamina epithelialis; LP, lamina propria; ID, intestine diameter; VL, intestinal fold length; MT, muscle layer thickness

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Liver samples were collected and processed in a tissue processor using the same histological protocol as intestine samples. Concerning liver slides, 15 microscopic fields per fish were taken using 400× magnification. From each field, three hepatocytes were randomly chosen for measurement, using the same methodology described previously. To avoid bias, only hepatocytes with a nucleolus in the focus were taken for evaluation (Rašković et al. 2019). Cell and nucleus surface area, nucleus circularity, and nucleus distance from the nearest cell membrane (LD) were evaluated for each hepatocyte.

All microscopic slides derived from samples were photographed using a Leica DM LS light microscope (Leica Camera AG, Wetzlar, Germany) with a DC 300 camera (Leica Camera AG). Images were analysed using ImageJ version 1.50e (Schneider et al. 2012).

Statistics

Data were tested for normality with the Shapiro-Wilk W test and analysed with one-way ANOVA and Mann-Whitney-U for non-parametric and Tukey`s pairwise test for parametric analyses. Statistical analyses were carried out using Past 3.17 (Øyvind Hammer, Natural History Museum, University of Oslo, Norway).

Results

Experimental conditions and fish growth

Water physical and chemical properties are shown in Table 2. From the beginning of the experiment in August to the end of the experiment in October, the mean water temperature dropped from 22 to 12 °C, and the mean oxygen concentration rose from 5.41 to 5.62 mg L-1. This change in temperature affected the metabolism of fish and their growth rate, and it was best reflected in a steady drop of SGR throughout the experiment (Fig. 3). After 15 days of the experiment, significant differences in BWG and SGR were noted (p < 0.05). As time elapsed, the differences between groups became more prominent. Fish fed extruded and pelleted diets showed no differences throughout the experiment for all three parameters, except BWG in the last 20 days. Meanwhile, the control group and wheat-fed group showed significantly lower performance (p < 0.05). Until days 45–60 they expressed similar values for all measured growth parameters. Later, values of SGR and CF of the group fed with wheat became significantly higher than those of the control group (p < 0.05). Differences in CF prevailed after one month, where C and E had the lowest and highest values respectively, while the other two groups were positioned intermediately (p < 0.05). These differences became wider at the end of the experiment.

Fig. 3
figure 3

Growth and fitness parameters dynamics measured at the end of every 15 days. a Body mass; b BWG, body weight gain; c SGR, specific growth rate; d CF, Fulton`s condition factor. All values are presented as means ± SD. a,b,c groups defined by statistically significant differences at p < 0.05. C, control group; W, wheat fed group; P, the group fed pelleted diet; E, group fed extruded diet

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Histological evaluation

Histology results in some way correspond to results of fish growth performance, where groups fed pelleted and extruded diets, in most cases, outstands other two groups.

Erythrocytes morphology

Erythrocytes from fish in the control group had the smallest mean surface area and lowest degree of circularity; their shape was the furthest away from the ideal circle (ideal circle represents a value of 1). On the opposite side are erythrocytes from fish fed extruded diet, and in between erythrocytes from fish fed with wheat and pelleted diet (p < 0.05). The longer axis was a major contributor to erythrocyte surface area differences, compared to the shorter axis, which was significantly larger only in E compared to the other groups (Table 3). Erythroblast count did not show any significant differences between groups.

Table 3 The intestine cross-section, erythrocytes and hepatocytes measurements.
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Morphology of hepatocytes

Hepatocytes showed a similar trend compared to erythrocyte morphology. The smallest mean hepatocyte surface area was present in the control group compared to all other groups (p < 0.05). The similar trend was observable in hepatocyte nuclei; nuclei from the control group were shaped in an almost ideal circle with the smallest surface areas. The only discrepancy from this trend was noted in measurements of nuclei distance from the nearest cell membrane. Here, nuclei from W group had a significantly larger distance from the cell membrane, compared to the other groups (Table 3).

Intestine histological morphology

Cross-sections of fish intestines were similar in size, except for those from the control group, which had a 15–20 % shorter intestine diameter (p < 0.05). Mean intestinal muscle layer thickness was similar in length in C and W groups and was almost half the thickness of P and E groups (p < 0.05). Mean intestinal fold length showed an almost linear increase between groups, in the following order: E > P > W > C.

Volume density, which the lamina propria and goblet cells cover in the intestine cross-sections, was statistically the same in all groups. The tunica mucosa was thicker only in the E group, while IEM volume density differed. C group had the lowest number of macrophages. W had a higher number of macrophages, and P and E were similar (Table 4).

Table 4 Results of intestine stereological measurements
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Discussion

Processed cereals, as well as pelleted and extruded formulated diets are well documented to have a high performance on fish growth (Booth et al. 2002; Przybyl and Mazurkiewicz 2004; Venou et al. 2009; Másílko et al. 2014; Ma et al. 2015; Hlaváč et al. 2015, 2016a, b; Marković et al. 2016). They are better balanced to meet carp feeding requirements compared to W and C. In our experiment, P and E diets differed slightly from each other, with a somewhat better performance of the extruded diet, namely higher BWG. In addition to the higher protein content in the extruded diet, there were two reasons that could explain this difference. During the extrusion process, the nutritional value of feed is increased due to the removal of heat-labile secondary compounds of plant-based feed components (Drew et al. 2007; Gatlin et al. 2007), which gives E a higher digestibility over P. The quality and quantity of lipids in feed had a profound effect on fish growth (Watanabe 1982). Various authors reported lower optimal values for common carp lipid requirements: 6 % (Abbass 2007), 8 % (Poleksić et al. 2014b), and 9 % (Ahmad et al. 2012). Thus, higher lipid content in P negatively influenced fish growth performance (Poleksić et al. 2014b). It seems that natural food was sufficient to obtain similar growth in the control group (C) as it was in the group that received wheat as an additional feed (W). There was no statistically significant difference between C and W groups in the total mass and BWG at the end of the experiment, but W had a slightly better SGR and CF. This difference can be explained by considering the reduced availability of zoobenthos, which is the preferred natural food of common carp (Rahman and Meyer 2009), which left fish in the C group mainly dependent on zooplankton as its sole food-source.

Fish that suffered a longer period of starvation tend to have significantly decreased erythropoiesis (Rios et al. 2005; Kondera et al. 2017). Immature erythrocytes in common carp are smaller in size and more round than mature erythrocytes (Witeska et al. 2010), but an opposing situation was noted. In the control group, circularity was the lowest. Similarly, mean cell size had the lowest circularity. Erythroblast frequency did not significantly differ among groups; with x̅ = 11.5 ± 5.2 %, they were within the standard values for juvenile common carp (Kondera et al. 2019). These shifts in erythrocyte sizes and circularity can be explained by prolonged erythrocyte life and slowed erythropoiesis in starving fish (Rios et al. 2005; Pronina and Revyakin 2015). On the contrary, the normal erythroblast frequency may represent haemolytic anaemia followed by higher erythropoietic activity in the malnutrition fish (Jain 1993). Judging from the growth performance, fish from the C group were not starving, but we can assume they suffered malnutrition to some lower extent. Their food intake was irregular and less diverse compared to other groups, as it was dependent only on invertebrates that entered cages.

The surface area of hepatocyte nuclei cross-sections were used as indicators of nutritive physiology in fish feeding experiments. This was demonstrated in other experiments where the same feeds were tested on common carp (Rašković et al. 2016a, b). Reduction in hepatocyte size is another indicator of malnutrition in group C, as Rios et al. (2007) and Park (2018) described it as a feature of fish that underwent starvation.

Poleksić et al. (2014b) showed that hepatocyte nuclei size was altered in feeds that contained a fat content higher than 8 %. In general, smaller nuclei in hepatocytes indicate reduced metabolic activity (Rios et al. 2007), which is supported with studies of fish malnutrition or starvation (Strüssmann and Takashima 1990; Margulies 1993).

The nucleus distance from the nearest point of the cell membrane was used as a direct measurement of nuclear displacement, which is an indicator of lipid reserves building up in hepatocytes (Caballero et al. 2004). The only significant higher nuclear distance was observed in hepatocytes from the W group, meaning that nuclei in this group were more centrally positioned, which implicates that built up reserves were lower compared to other groups. This is followed by the lowest nucleus/cell surface ratio coupled with the highest mean cell size in W. At the end of this experiment, we analysed for the presence of fat in the liver. Samples were frozen in liquid nitrogen, cut on a cryotome, stained with oil-red O staining, and examined under the microscope. All samples were negative on lipids. This follows our previous results, where fat disappeared from carp livers in the autumn season (Rašković et al. 2016a). In addition, there were no hydropic degeneration present in the hepatocytes; therefore, we can conclude that the build-up of reserves were most likely glycogen.

Intestine morphology can be modulated by feed ingredients; in that manner, intestine morphology reflects its capability for food digestion and nutrient utilisation (Baeverfjord 1996; Klurfeld 1999; Khodadadi et al. 2018). The length of intestinal folds was  positively correlated with nutrient absorption, therefore affecting fish growth (Farhangi and Carter 2001; Zhou et al. 2010). Bakhshi et al. (2018) reported intestinal fold shortening in common carp fed on biofloc with the addition of sugar and corn starch. Rašković et al. (2015, 2016a) observed a positive correlation between common carp body weight and intestinal fold length. Intestinal fold length was E > P > W > C, but the intestinal fold length, together with other size-related features, should be compared only between C and W groups and P and E groups. The reason is that there were no statistically significant differences in mean body mass of fish from C compared to the W group and P compared to the E group. Higher intestinal fold length and higher whole section diameter were observed in fish fed W compared to C group. In addition, there were no differences between these two groups for the following features: muscle layer thickness, lamina propria, and lamina epithelialis volume densities. When compared, E and P groups had a similar trend. There were no differences in whole section diameter size, muscle layer thickness, lamina propria, and lamina epithelialis volume densities, but intestinal fold length was significantly higher in the E group. Here, we hypothesised that fish from the C group to some extent experienced atrophy of the alimentary tract (smallest intestine diameter and fold length together with reduced muscle layer thickness) due to malnutrition. The rationale for this claim is that despite common carp is primary benthivore fish, in the control group it still could meet some of its nutritional needs by grazing on zooplankton. Lack of mortality increment between groups supports claim that fish did not starve, but malnutrition was possible. In addition, fish experience proteolysis of the intestinal mucosa in the short-term (Ostaszewska et al. 2006) and decrease their body mass in long-term starvation (Rios et al. 2002; Rios et al. 2006), which was not the case in the present study. In terms of histological structure, natural feed is the optimal choice for fish feeding, which was proven in several laboratory trials (Kamaszewski and Ostaszewska 2014; Ostaszewska et al. 2018). However, if fish are fed unsuitable compound feed, it would also show severe pathologies in both the liver and intestine, and as a result, fish would experience atrophy of the intestinal tissue (Kasprzak et al. 2019).

Macrophages are the first line of defence in the fish innate immune system, as they phagocytise foreign particles (Ellis et al. 1976). The highest number of IEM in fish fed an extruded diet corresponds to data reported by Poleksić et al. (2014a). The macrophage number increases in fish intestines, starting from the control group, which may indicate that additional feeds contained an increased number of particles that may be identified as antigens by the fish immune system. These particles may originate from chemicals used in feed production, such as pesticides, or from storage, as protein rich feeds are more prone to the development of microorganisms. In addition, formulated feeds contain soybean meal. Soy contains anti-nutritive factors that are known to induce distal intestine inflammation in fish (Urán et al. 2008). This inflammatory response is characterised by the presence of immune cells common for both a specific and non-specific immune response (Krogdahl et al. 2010).

Shortening of the intestinal folds, together with lamina propria thickening and leucocytes infiltration in the lamina propria and submucosa, are some of the symptoms of distal intestine alterations (Heikkinen et al. 2006; Refstie et al. 2000, 2001). An increase in the number of IEM alone is not enough to claim intestine inflammation but may bear some concerns for a health risk.

Conclusions

Additional feeds have a substantial influence on fish nutritional physiology. Judging from the histological effects on common carp liver and intestine, morphology of erythrocytes, and growth parameters, the extruded feed may be the best choice, but it raises some concerns over the fish physiological status due to the higher number of IEM. In contrast, wheat had no significant effect on the increase of IEM, but all other parameters were low. Growing carp in a cage system is a rare practice and it relies on additional feeds. We can conclude that if common carp feeds on naturally occurring zooplankton, this can contribute to its growth, but without additional feeding on benthos organisms or given feed, fish will probably suffer from malnutrition issues. In conclusion, feeding 3 % of the total mass with pelleted feed daily may be the best choice for common carp yearlings, judging from the perspective of growth performance and fish welfare. In addition to feed price and growth performance, different additional feeds may have different effects on the distal intestine, liver, and red blood cells, which can be important when it comes to feed choice in carp nutrition. More long-term feeding trials are necessary to draw more accurate conclusions on feed choice in carp nutrition.