Introduction :

Protein content in fish feed is one of the most important nutrients. Protein levels in the diet significantly affect growth rates, feeding efficiency, and tissue health in fish [1] . Azolla plant is a small, floating aquatic plant that grows rapidly and covers the surface of ponds during the rainy season. It serves as a free food source. A member of the Salviniaceae family, it can be used as a direct fish feed or as a feed ingredient, providing an alternative protein source.It is a low-cost, locally available plant [2] [3]. It is rich in amino acids such as arginine, valine, lysine, and leucine, as well as a rich source of unsaturated fatty acids [4]. The grass carp recently reclassified in the family Xenocypridinae rather than Cyprinidae based on recent evolutionary studies [5], prefers to feed on aquatic plants, including Azolla Pinnata. It has been shown that adding plant protein can improve meat quality by enhancing muscle characteristics, reducing fat accumulation, and boosting collagen production [6] [7]. Studies have also demonstrated that providing optimal protein levels in fish diets increases muscle protein concentration and improves gill function [8]. The current study aims to determine the effect of different concentrations of Azolla in the diet on protein concentrations in the gills and muscles of grass carp fingerlings.

Materials and Methods

2.1 Sample collection

120 samples of grass carp fingerlings (C. idella) were collected from the fish farming ponds of the Marine Science Centre/University of Basra between December 2024 and January 2025. The samples were transported using special plastic containers to the Environmental Laboratory of the Department of Life Sciences at the College of Education, Al-Qurna/University of Basra. The fish were acclimatised for ten days. They were then randomly distributed into six glass tanks with dimensions of 40cm x 60cm x 40cm as follows:

1. The two control groups (T1): Each group consisted of 20 fish fed a conventional diet free of Azolla.
2. The two 15% replacement groups (T2): Each group consisted of 20 fish fed a diet containing 15% Azolla.
3. The two replacement groups (T3) (30%): Each group consisted of 20 fish fed a diet containing 30% Azolla.

During the experiment, the fish were fed a laboratory-formulated diet at the studied proportions (Table 1).

2.2 Protein Extraction

Protein was extracted in the studied areas. Total protein was extracted using the method of [8], which involves: taking 0.5 g of the sample and crushing it using a mortar; adding 1 ml of the protein extraction buffer prepared previously at the Iraqi Biotechnology Company/Molecular Biology Laboratory (Table 2); mixing the samples thoroughly; and storing the mixture in a 1.5 ml Eppendorf tube. The mixture was left to stand for 30 minutes at -4°C. The mixture was then centrifuged at 16,000 rpm for 10 minutes. The top layer of the separated liquid was transferred to a new tube of the same capacity and stored at -20°C until use in protein concentration determination.

2.3 Protein Concentration Estimation

The protein concentration in the gills and muscles of the studied fish was determined using the Bisinchoninic Acid Assay (BCA). A special kit from Thermo Fisher Scientific was used, following these steps:

1. Prepare the working solution (WR) by mixing 50 parts of reagent A with 1 part of reagent B (1:50 ratio of reagents B: A).
2. Prepare different concentrations of known proteins, such as bovine serum albumin.
3. Add 25 µL each of the standard solution and the protein samples of unknown concentration to the wells.
4. Add 200 µL of the reagent to each well and mix thoroughly using a plate shaker for 30 seconds.
5. Cover the plate with a plate stopper and incubate for 30 minutes at 37°C, then cool to room temperature. 6- Measurement of wave absorption at a wavelength of 562 nm using a device ( ). The protein concentration in the muscles of the two regions and the gills was determined according to the curve in Figure 1.

Figure 1. Figure 1: Standard curve for estimating protein concentration

3. Statistical Analysis

Statistical analyses were performed using IBM SPSS Statistics 22. The data were analyzed using one-way analysis of variance (ANOVA), followed by Fisher's Least Significant Difference (LSD) test for multiple comparisons. Differences were considered statistically significant at p < 0.05.

4. Results and Discussion

Aquatic plants are an important food source for herbivorous fish, including grass carp. Plant-based protein, such as that derived from Azolla sp., can replace a significant portion of the expensive animal protein used in commercial feeds. This replacement can reach up to 50% of the feed components without negatively impacting growth or body tissue development [9]. Protein concentration in different body tissues of fish is affected by several factors, most importantly nutrition, season, and physiological state [10]. Therefore, three different percentages of Azolla sp. (15%–30%) were used in this study to determine the effect of these percentages on protein concentration in certain body tissues (gills and muscles) of grass carp fingerlings.

Therefore, the results of the current study showed clear effects on protein concentration values ​​in the gills of the studied fish. An increase in gill protein concentration was recorded during the first time period, from 5492 mg/ml to 6035 mg/ml in treatment t3. Despite this difference in protein concentration levels, no significant differences (P>0.05) were observed (Figure 2A). In the second period, the protein concentration was recorded at 6703 mg/ml in T1 and 5437 mg/ml in T3. In T3, the gills showed a protein concentration of 6938 mg/ml. This difference in protein concentration levels led to significant differences (P>0.05) (Figure 2B). Meanwhile, during the third time period, the protein concentration increased to 7276, 8331, and 8519 mg/ml in T1, T2, and T3, respectively (Figure 2C). This resulted in statistically significant differences (P<0.05). The variation in gill protein concentrations ​​indicates that adding Azolla to fish feed may affect gill protein levels. Previous studies have shown that these effects are related to several factors, including concentration, water type, and target fish species [11]. This suggests that Azolla may improve water quality by adsorbing pollutants and providing a healthier environment for fish, thereby promoting better growth and health [12]. This reflects the quality and effectiveness of the feed in supporting vital functions, particularly enhancing the immune system. Since gills are the primary lines of defence for fish, increased protein concentration in them is an indicator of healthy, intact, and strengthened gill tissue [11].

Muscle tissue constitutes 60-80% of the fish's body and is divided into two main types: red muscle and white muscle. These two types are classified based on colour, appearance, location, blood supply, certain biochemical properties, and histological characteristics related to the number and diameter of red and white muscle fibres [13] [14]. Muscles are among the richest sources of protein in the body, reflecting the presence of essential and non-essential amino acids, which play a crucial role in muscle building and growth [15]. They also have high biological value [16], containing approximately 15-25% protein. The protein content of muscle tissue is the second most abundant component after moisture in fish [17] [18]. Using protein-rich Azolla in varying proportions as a substitute for feed ingredients, the current study showed that this plant increases protein concentration in the R1 and R2 anterior muscles of the studied fish. The results showed an increase in protein concentration in the R1 anterior muscles (8940, 7655, 7757 mg/ml) in T1, T2, and T3, respectively (Figure 3B). Protein concentration values ​​of 9729, 9939, and 8486 mg/ml were recorded in T1, T2, and T3, respectively (Figure 3A). Statistical analysis of the results showed significant differences (P<0.05) for the R1 and R2 anterior muscles (Figures 3 and 4). The current results also showed an increase in protein concentration in the anterior muscles during the second period, reaching 8404, 6698, and 5998 mg/ml in T1, T2, and T3, respectively (Figure 3).

Meanwhile, the protein concentration in the posterior R2 muscles was 7750, 7833, and 6673 mg/ml in T1, T2, and T3, respectively (Figure 4). As a result of these differences in protein concentration levels in R2 and R1, the statistical results showed significant differences (P<0.05) (Figures 3 and 4). The current results also recorded an increase in protein concentration in the R2 and R1 muscles during the third period, with protein concentrations of 8884, 8466, and 7358 mg/ml in the anterior muscles in treatments T1, T2, and T3, respectively (Figure 3). In the hindquarters (R2), protein concentration ranged from 8650 to 8000 mg/mL during T1, T2, and T3, respectively (Figure 4). Analysis of protein concentration results during the third period revealed significant differences (P<0.05) (Figures 3 and 4). This increased protein concentration in the hindquarters compared to the forelegs is attributed to the functional role of the red and white skeletal muscles in the posterior region of the body and their contribution to locomotion in fish. The muscles of the posterior region, along with the caudal fin, form the primary locomotion organ in fish [19].

Additionally, the inclusion of azolla in the fish diet may contribute to this effect. Previous studies have shown that adding azolla to fish feed increases the protein content of fish muscle. Azolla is a good food source, meeting most of the protein and essential amino acid requirements of herbivorous fish [20]. Incorporating protein into fish feed can positively or negatively affect protein concentration in the gills and muscles of fish. This depends on several factors that affect protein concentrations in the gills and muscles, including the type and quantity of Azolla used, the type of fish, and environmental factors that affect the fish's diet [21] .

Figure 2. Figure 2 . The effect of treatment with different concentrations of A. pinnata on the protein concentrations in gills. The various letters refer to significant differences

Figure 3. Figure 3 . The effect of treatment with different concentrations of A. pinnata on the protein concentrations in R1. The various letters refer to significant differences

Figure 4. Figure 4 . The effect of treatment with different concentrations of A. pinnata on the protein concentrations in R2. The various letters refer to significant differences

Conclusion

The study concludes that there is a clear variation in protein concentration rates in the studied body regions, with an increase in protein concentration correlating with the rise in the percentage of Azolla in the diet. These differences are evidently due to the effect of feeding and the replacement level, in addition to other factors.