Abstract 
In the present study, the effects of different levels of alfalfa powder and alcoholic extract on the activity of digestive enzymes and liver compounds of common carp (Cyprinus carpio) juveniles were examined. For this purpose, 270 fish (with an average initial weight: 20.04±0.19 g) were randomly distributed in 27 polyethylene tanks and fed by the carp commercial diet. The experimental treatments of 9 groups (4 groups with 3, 6, 9, and 12% of alfalfa powder, 4 groups with 1, 2, 3, and 4% of alcoholic extract and one control group) with three replications were considered for 8 weeks. At the end of the experimental period, the activity of digestive enzymes was measured in terms of U/mg Protein and liver composition. Based on the results, the highest levels of activity of digestive enzymes, amylase (674.74±61.57), lipase (6.21±0.96), alkaline phosphatase (452.84±28.18), trypsin (252.05±19.98) and chymotrypsin (0.10±0.001) in the extracts of 3%, 3%, 3%, 4% and 4% extracts, respectively, and the lowest amount of these enzymes were observed respectively with values of 388.17±38.27 (control), 2.51±0.55 (control), 187.7±19.36 (12% powder), 0.030±0.004 (12% powder) and 132.47±11.47 (9% powder) (P <0.05). The highest amount of hepatic glycogen (3383.35±192.13 µg/g tissue) was measured in the 3% extract treatment and the lowest amount of hepatic glycogen (607.01±101.23 µg/g tissue was in the control treatment (P <0.05). The highest level of liver lipids (146.51±6.05 mg/g tissue) was observed in the treatment of 6% powder and the lowest level of liver lipids (72.99±4.46 mg/g tissue) was observed in the treatment of 12% powder (P <0.05). According to the present study, adding powder and especially alfalfa extract to the diet of common carp can increase the activity of digestive enzymes and increase liver lipid and glycogen at certain levels. A diet containing 3% alfalfa extract had a significant effect on the activity of digestive enzymes and liver glycogen levels. Also, a diet containing 9% alfalfa powder significantly increased the activity of lipase enzyme and liver lipid levels. According to the results, the use of alfalfa extracts up to a level of 3% in the diet of common carp, as a growth supplement and nutrition are recommended.
1. INTRODUCTION
Improving feed efficiency in aquaculture is a critical factor in reducing production costs and enhancing growth performance, as a substantial proportion of total expenses is associated with feed. In recent years, increasing attention has been directed toward the use of natural and plant-based additives, particularly phytobiotics, as safe alternatives to synthetic growth promoters. Digestive enzyme activity and nutrient absorption play a crucial role in providing a stable energy supply and promoting fish growth, as higher secretion and activity of these enzymes enhance nutrient availability and efficient digestion (Blier et al., 1997; Nya and Austin, 2011; Awad et al., 2012). Medicinal plants such as alfalfa contain bioactive compounds, including flavonoids, alkaloids, and essential fatty acids, which can serve as growth promoters, appetite stimulants, and enhancers of digestive enzyme activity in aquafeeds (Sivaram et al., 2004; Yu et al., 2008). Previous studies have demonstrated that supplementation of fish diets with alfalfa extract can significantly increase the activity of amylase, lipase, trypsin, and chymotrypsin, thereby improving digestion and nutrient utilization (Zhang et al., 2009; Kolivand, 2020; Falamarzi et al., 2016). Given that common carp (Cyprinus carpio) is one of the most important cultured fish species worldwide and that digestive enzyme activity and hepatic metabolism play crucial roles in its growth, the present study aimed to evaluate the effects of dietary alfalfa powder and extract on digestive enzyme activity and liver glycogen and lipid levels in juvenile common carp during an eight-week feeding trial.
2. MATERIALS AND METHODS 
An eight-week feeding trial was conducted to evaluate the effects of alfalfa (Medicago sativa) powder and ethanolic extract on digestive enzyme activity and liver biochemical composition in juvenile common carp (Cyprinus carpio; 20.04 ± 0.19 g). A total of 270 fish were randomly distributed into 27 tanks (10 fish/tank) and acclimated for two weeks. Nine dietary treatments, including graded levels of alfalfa powder (3–12%) and extract (1–4%), along with a control, were formulated from a commercial carp diet. Fish were fed to satiation three times daily, with water quality monitored throughout the trial. At the end, intestinal and liver samples were collected to measure enzyme activities (amylase, lipase, trypsin, chymotrypsin, alkaline phosphatase) and liver glycogen and lipid content using standard biochemical assays. Data were analyzed using one-way ANOVA and Duncan’s post hoc test. This study provides a controlled evaluation of alfalfa as a phytobiotic feed additive, highlighting its potential to enhance digestive efficiency and liver metabolic status in common carp.
3. RESULTS 
The effects of different levels of alfalfa powder and ethanolic extract on digestive enzyme activities and liver composition in juvenile common carp (Cyprinus carpio) were evaluated at the end of the feeding trial. Digestive enzymes measured included amylase, lipase, alkaline phosphatase, trypsin, and chymotrypsin. The highest amylase activity was observed in treatment E3 (57.74 ± 61.674 U/mg protein), while the control exhibited the lowest (27.17 ± 38.388 U/mg protein). Lipase activity peaked in treatments E3 and P9, whereas the control had the lowest value. Alkaline phosphatase activity was highest in E3 and lowest in P12. Trypsin and chymotrypsin activities reached their maximum in E4, while the lowest activities were observed in P9, P12, and the control. Liver biochemical analysis showed that glycogen content was highest in E3 (192.3383 ± 13.35 µg/g tissue) and lowest in the control, with significant differences between treated groups and control. Liver lipid content was highest in P9 (146.6 ± 5.51 mg/g tissue) and lowest in P12, with significant differences among treatments. These results indicate that specific levels of alfalfa supplementation can enhance digestive enzyme activity and liver energy reserves in juvenile common carp.
4. DISCUSSION AND CONCLUSION 
Supplementing juvenile common carp diets with alfalfa, especially as extract, enhanced digestive enzyme activities (amylase, lipase, trypsin, and chymotrypsin), improving carbohydrate and protein digestion. Alfalfa extract increased liver glycogen, likely via enhanced glucose absorption and insulin stimulation, while liver lipid content was moderately affected, correlating with lipase activity. Powdered alfalfa showed weaker or sometimes inhibitory effects, probably due to anti-nutritional factors. Overall, alfalfa, particularly as extract, improved nutrient utilization and energy storage, supporting growth in common carp. Adding alfalfa powder and especially extract to the diet of common carp, particularly at 3% extract, increased digestive enzyme activities and liver reserves (glycogen and lipids), which can enhance growth and nutritional indices; thus, using alfalfa extract up to 3% of the diet is recommended.

ACKNOWLEDGEMENT 
The authors of this paper express their sincere gratitude to the Research and Technology Deputy of Khorramshahr University of Marine Science and Technology for their financial support and for providing the necessary equipment for this study.
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