Introduction
For animal-based products, including aquatic products, appearance characteristics play a critical role in influencing consumer preferences.1,2 The pigments present in the feed serve as the primary source for the development of body coloration in aquatic animals. The main types of feed pigments include carotenoids, pterin, purine, and melanin.3 Carotenoids are important coloring substances in aquatic products.4 However, fish cannot biosynthesize carotenoids but rely entirely on natural or synthetic pigments.5 The use of natural pigments in aquaculture instead of synthetic pigments is beneficial to public health and safety.6
The gut microbiota plays a crucial role in the healthy development of fish by regulating gastrointestinal development, supporting nutrient metabolism, and enhancing immune response.7–9 Feed is the main factor affecting the composition of fish gut microbiota. The active ingredients in the feed can regulate the intestinal flora and improve the balance of gut microecology. Previous studies have shown that lutein or ferrous fumarate can enhance stress resistance, digestive capacity, and promote growth.10,11 Lutein is an important component of carotenoids, and like other carotenoids, it cannot be synthesized in the human and animal body. Therefore, it needs to be obtained from food. Lutein is widely distributed in natural plants and serves as an important substance for fish coloration. Additionally, lutein also possesses antioxidant activity, which can effectively improve animal health.12 Ferrous fumarate is a safe and effective organic nutritional iron supplement that can improve the anti-stress and anti-disease ability of animals, has a good synergy with various nutrients, and has the effect of promoting animal growth and enhancing immunity.10
To date, little information about the effect of lutein and ferrous fumarate on common carp has been reported. Common carp (Cyprinus carpio) is an important species in global aquaculture, with a global production exceeding 4.1 million tons in 2017 (FAO 2019). The Yellow River carp (Cyprinus carpio), as a local variety of carp, is mainly distributed in the Yellow River basin of China and is famous for its tender and tasty meat, long body shape, golden scale, and red tail, and valuable fish resource in China. However, intensive aquaculture practices have negatively impacted the growth performance of Yellow River carp, particularly in terms of coloration, flesh quality, and disease resistance. These factors seriously constrain the production and cultivation of carp. Lutein, being a rich source of bioactive compounds, combined with the significant biological activity of ferrous fumarate, presents a promising candidate for use as a functional feed additive. In the study, the hypothesis was that the supplement of lutein and ferrous fumarate to carp diets would lead to beneficial effects on skin color and other physiological functions of Yellow River carp. In this regard, this study aimed to evaluate the effect of lutein and ferrous fumarate on the pigmentation, digestive enzyme activity, and gut microbial community of Yellow River carp.
Materials and methods
Experimental fish and diet preparation
Yellow River carp was provided from a carp culture farm in Luoyang city, China. The carp were then disinfected with sodium chloride (2%, 15 minutes) and placed in five 500-liter tanks (2 weeks) for acclimation. During the two-week acclimatization, fish were fed with the control diet. At the beginning of the experiment, 135 carps (25.0±2.0g) were weighed and randomly distributed into three experimental treatments with three tanks (15 fish in each tank, 100 × 80 × 60 cm) for the 6-week experiment. During the experiment, carp were fed at 3% of their body weight twice a day at 8:30 and 14:30, respectively, weighed once every two weeks, and then adjusted the feed, and the water in the tank was continuously aerated and changed regularly. Water temperature (22-25℃), dissolved oxygen (DO > 5 mg/L), pH (7.1-7.4), and NH3-N < 0.4 mg/L were monitored every day by a Water quality analyzer (Genesite, China).
Three different diets were designed to feed experimental fish: the basal diet (control group), feed with 150mg/kg lutein, feed with 150mg/kg lutein and 100mg/kg ferrous fumarate. The lutein and ferrous fumarate were both from Chenguang Biology Co., Ltd. (China), the purity of lutein is 90 %, and the purity of ferrous fumarate is 95 %. Commercial carp feeds purchased from Tongwei Co., Ltd. (China) were powdered, the test raw materials were added, mixed with a small mixer, and finally, 2 mm granules were made with a micro granulator. Dried feeds were stored at -20°C.
The fish were fasted for 24 hours before sampling at the end of the experiment. The weight of the experimental fish was measured and recorded, the number of fish was counted, and the survival rate and specific growth rate were calculated. In addition, three randomly selected carp were taken from each replicate tank and treated in a diluted MS-222 solution. Organs were separated and weighed, viscerosomatic index (VSI), hepatosomatic index (HSI) and Intestosomatic index (ISI) were calculated. Then, the small intestine was quickly frozen in liquid nitrogen and stored at -80°C for further examination of intestinal microbiota, which was sent to Shanghai Yuanshen Biotechnology Co., Ltd. for analysis. The foregut was used for the determination of digestive enzyme activity. Digestive enzymes, including intestinal amylase (AMS), lipase (LPS), and trypsin (TPS), were measured using the reagent kit provided by the Nanjing Institute of Bioengineering Research. The kits included an α-amylase assay kit (C016-1-1), a lipase assay kit (A054-1-1), and a trypsin assay kit (A080-2-2). Additionally, the skin and scales were dissected, washed, and drained to determine the content of carotenoids. The determination of carotenoid content in fish followed the method described by Torrissen and Naevdal.13
Skin pigmentation
At 0 d, 15th d, and 42th d of the experiment, the L*, a*, b* of carp skin color were measured near the lateral line and back according to CIE2000 L*a*b* color system with colorimeter of Konica Minolta CR 400. CIE2000 L*a*b* color system according to Hunt14 and Berns.15
Intestinal microbiome analysis
After the experiment, samples of intestinal contents collected from the fish tank were processed using the QIAamp DNA Stool Mini Kit from Qiagen, Germany. Microbial DNA was extracted from the samples. The extracted microbial DNA was then subjected to PCR amplification using universal primers targeting the V3-V4 variable region of the 16S rRNA gene. These primers are known for their high conservation and variability in the V3-V4 region, which allows for identification and description of microbial community composition and structure. The PCR amplicons were analyzed using version 1.17 of the Qiime (Quantitative Insights Into Microbial Ecology) toolkit. By processing and analyzing the raw read data, further insights into the composition, diversity, and functional characteristics of the microbial community in the fish tank can be obtained.
Statistical analysis
All results are presented as mean ± SEM. Significance was determined using one-way analysis of variance (ANOVA) and Tukey’s post hoc test. When the P value was 0.05, the groups were significantly different. Statistical analysis was performed using SPSS v17.0 (SPSS Inc., Chicago, IL, USA).
Results
Growth performance
During the 6-week experiment, no significant difference in final weight, hepatosomatic index, and specific growth rate was observed among treatments (Table 1). In addition, compared with the control group, the viscerosomatic index and the intestosomatic index value were significantly increased in the lutein and ferrous fumarate group compared to the control group (P < 0.05). No mortality was observed during the experimental period.
Skin pigmentation
As can be seen from Fig.1, after feeding the lutein and ferrous fumarate for 42 d, the carp showed different color changes, with the total carotenoid content in carp significantly increased (P < 0.05). The L* (light) value of the control group was the highest on the 15th day and the 42nd day compared to the experimental treatments (P < 0.05). However, the b* (blue), Ch* (chroma) and ΔE (Color difference) values of carp fed with lutein or ferrous fumarate were significantly higher than that of the control group (P < 0.05) (Table 2).
Digestive enzymes activities
The activities of digestive enzymes in the foregut of Yellow River Carp are provided in Table 3. The highest amylase, lipase and trypsin activity were observed in the supplemented diet with lutein and ferrous fumarate. No significant difference was observed among experimental treatments (P > 0.05).
Intestinal microbiota
As shown in Fig.2, Venn diagrams illustrated the distribution of OTUs in experimental samples. Three groups shared 153 OTUs. Additionally, 99 unique OTUs are found in the lutein group, indicating their sensitivity to lutein. Furthermore, 21 unique OTUs are found only in the lutein and ferrous fumarate mixed group, suggesting their sensitivity to ferrous fumarate.
As shown in Fig. 3, after being fed with the lutein and ferrous fumarate, at the phylum level, the dominant phyla of intestinal microbiota in carp are Proteobacteria, Actinobacteria, and Bacteroidetes. Compared to the control group, the abundance of Proteobacteria is lower, while the abundance of Actinobacteria is significantly increased (P < 0.05). As illustrated in Fig. 4, at the genus level, compared to the control group, the abundance of families such as Rhodobacteraceae and Microbacteriaceae was significantly decreased in the group with lutein and ferrous fumarate (P < 0.05), while the genus Flavobacterium is significantly increased (P < 0.05).
_relative_abundance_of_intestinal_microbiota_at_the_phylum_level._(b)_abundance_differenc.png)
_relative_abundance_of_intestinal_microbiota_at_the_genus_level._(b)_abundance_difference.png)
Discussion
The research findings showed that the addition of lutein and ferrous fumarate in feed can promote the specific growth rate, body weight, and total carotenoid content in the skin and scales of carp. Some intermediate metabolites of ferrous fumarate and lutein in the feed may be the reasons for the improvement in the growth performance and viscerosomatic and intestosomatic index of the experimental carp. Previous studies have shown that lutein can promote feed utilization and growth performance of fish, while ferrous fumarate can enhance stress resistance, digestive capacity, and promote growth.10,11,16,17 Additionally, ferrous fumarate is an important trace element for the healthy development and physiological functions of fish. Research has shown that the addition of ferrous fumarate can effectively improve the composition of the intestinal microbiota in Yellow River carp, promoting the reproduction and metabolic activity of beneficial bacteria, thereby enhancing digestive function and immune capacity.10
Fish cannot synthesize carotenoids on their own and must obtain them from their diet.18 Therefore, it is commonly believed that adding carotenoids to feed is widely seen as one way to increase the sales of farmed fish.19,20 Body color as an important feature of fish can be evaluated by the L*, a*, b* values. In this study, the L* value representing the brightness of skin color was significantly lower in the experimental group compared to the control group at the end of the experiment. The experimental group of carp showed a nearly 2-3 times higher b* value, indicating a significant yellow color. Previous studies have also reported an increase in b* value in ornamental fish fed with natural or synthetic sources of carotenoids, which is consistent with the results of this study.21–23 Specifically, lutein is a natural compound that regulates the formation of skin pigmentation in fish. It has been found in the study that the addition of an appropriate amount of lutein can enhance the skin color of Yellow River carp, making it more vibrant and beautiful. This is because lutein is ingested by fish through food and accumulates in the skin, thereby altering their pigmentation. Body color is an important aspect of fish quality. It can be seen from the research results that adding a certain amount of lutein and ferrous fumarate to the feed can improve the effect of Yellow River carp, significantly increase the market acceptance of Yellow River carp, and at the same time, improve the breeding efficiency of Yellow River carp, while providing certain guidance for production.
Digestive enzymes in animals are located within the gastrointestinal lumen and are associated with the brush border membrane of intestinal epithelial cells.24 However, the digestive and metabolic functions of fish are obviously related to the enzyme activity of brush border.25 Digestive enzyme activity and species are critical factors influencing the absorption and utilization of nutrients in the gastrointestinal tract of animals. These parameters serve as valuable indicators for assessing the digestibility of fish.26 Feed composition can affect the digestive enzyme activity of aquatic animals. In this study, adding an appropriate amount of lutein or a mixture of lutein and ferrous fumarate to the feed effectively improved the activities of digestive enzymes such as amylase, lipase and trypsin in the intestinal tract.
Fish typically possess short digestive tracts, making their gut microbiota highly susceptible to external environmental factors. The composition of the gut microbiota in animals could affect their physiological status, living environment, and feeding habits. In addition, the diversity of the intestinal microbial community can improve the stability of the intestinal environment and enhance stress resistance. In the present study, according to the analysis of the Venn diagram, it was found that there are 99 unique OTUs in the lutein group, which may suggest that these specific OTUs may be sensitive to lutein. On the other hand, 21 unique OTUs were found in the carp fed with lutein and ferrous fumarate, indicating that these 21 OTUs may be sensitive to ferrous fumarate. In summary, the above results suggest that lutein or ferrous fumarate may have a positive impact on the survival and proliferation of intestinal microbiota. At the same time, these research findings indicate that lutein and ferrous fumarate have different mechanisms of action and impact on regulating the gut microbiota. However, the specific mechanisms and details still require further research and exploration for clarification. In this study, the addition of lutein or ferrous fumarate to carp significantly altered the composition of the intestinal microbiota at the phylum and genus levels. The lutein group exhibited a significant increase in the abundance of Bacteroidetes. This bacterial group is known to produce lipopolysaccharides (LPS), which are associated with inflammation and compromised intestinal barrier function. These findings suggest that lutein may exert a beneficial influence in mitigating these effects.27,28 Furthermore, the study also found that the abundance of Actinobacteria in the lutein and ferrous fumarate group was significantly increased. Actinobacteria has been shown to improve immune function and enhance disease resistance in fish. Increasing the abundance of Actinobacteria, lutein, and ferrous fumarate may contribute to the improved health status of carp.29,30 Meantime, after feeding the lutein and ferrous fumarate to carp, the abundance of Flavobacterium genus was significantly increased. These results suggest that lutein and ferrous fumarate can alter gut microbiota abundance. This suggests that the active substances in lutein and ferrous fumarate can influence the development of the gut microbiota. Lutein and ferrous fumarate can improve the intestinal microflora of Yellow River carp and increase the number of beneficial bacteria, thereby improving the intestinal environment of Yellow River carp, promoting its healthy growth, enhancing growth, and improving disease resistance. At the same time, it may also promote the coloration of Yellow River carp, which needs further in-depth research. Additionally, the study also documented the presence of some unknown microorganisms in the intestinal tract of the experimental carp. Further in-depth research on these unknown microorganisms can help us better understand their functions and potential impacts.
Conclusion
In conclusion, lutein and ferrous fumarate can effectively improve the skin pigmentation and intestinal microbial composition of Yellow River carp, thereby enhancing the coloring effect and digestive function. These findings provide valuable insights for optimizing feed formulation and aquaculture management, which can contribute to improving the quality and farming efficiency of Yellow River carp.
Acknowledgements
This work was supported by Henan Natural Science Foundation (182300410032) and Innovation and Entrepreneurship Training Program for college students in Henan Province (S201910464053).
Competing of Interest
The authors declare that they have no competing interests.
Authors’ Contribution
Conceptualization: Hongtao Ren (Lead). Formal Analysis: Hongtao Ren (Lead). Writing – original draft: Hongtao Ren (Lead). Writing – review & editing: Hongtao Ren (Lead). Data curation: Xiang Cao (Equal), Xiangzhi Guo (Equal), Peng Yuan (Equal). Investigation: Xiang Cao (Equal), Xiangzhi Guo (Equal), Peng Yuan (Equal).
Ethical Conduct Approval
This study was conducted under animal care approved by the Institutional Animal Care and Use Committee of Henan University of Science and Technology (No. 2021002).
Informed Consent Statement
All authors and institutions have confirmed this manuscript for publication.
Data Availability Statement
All are available upon reasonable request.