For sustainable aquaculture practice, use of cost-effective and alternate plant protein sources in replacement studies is still on its infancy. The probable causes of considering non-conventional plant feedstuffs not suitable as feed ingredient for fishes include meager nutritive potential in terms of poor digestibility and presence of anti-nutritional factors (Li et al. 2021). Cruz-Velasquez (2014) pointed out that aquatic macrophytes are imperative nutritional sources for herbivorous-omnivorous fishes and could replace 25% of formulated diets and 50% of commercial feeds without any detrimental effects on growth and body composition of fishes. The aquatic fern Azolla proliferate at high rates in freshwater bodies have modest protein content (19–31%) depending on their sources, strain and growing media (Fasakin et al. 2001). Water fern has gained attention as natural sources of food for fish species, either directly in fresh forms or in combination with other feedstuffs. Our results reveal the inadequacy of total replacement of fish meal with water fern at high levels of incorporation in practical diets for C. carpio communis fingerlings. The results of the present study are in conformity with the findings of El-Sayed, (1992) and Almazan et al. (1986) who reported that growth of fish decreases with increasing levels of added aquatic plant ingredients. Fasakin et al. (1999) also reported similar growth retardation and poor FCR when increased levels of dried water fern and duckweed meals were incorporated in the Nile tilapia diets. In the present study, it was evident that Azolla could be a good substitute for fish meal, where the replacement up to 10% of fish meal has no effect on growth performance of C. carpio communis fingerlings. Earlier reports suggested that inclusion of 25% Azolla microphylla and A. pinnata mixture in the diet of Labeo rohita significantly improved the growth and SGR (Datta 2011). Similarly in our study, incorporation of fish meal along with Azolla meal satisfied the nutritional requirement of C. carpio fingerlings. However, reduced feed efficiency at higher inclusion of Azolla meal (D5 (40) and D6 (50)) may be because of presence of anti-nutritional factors (ANFs) and high dietary fiber content which in turn reduced the growth performance (Kamali-Sanzighi et al. 2019; Magouz et al. 2020). It has been reported that higher dietary Azolla inclusion reduces the weight gain by increasing both the metabolic rate and energy expenditure, while decreasing the digestibility of ingredients, due to its ANF content (Ahmed et al. 2017; Mohammadi et al. 2018; Magouz et al. 2020).Nevertheless, the study of chemical and amino acid composition of water fern basically displayed the nutritional characteristics of these ingredients as fish feed (Chakrabarti et al. 2018).
Azolla can be used as a potential replacer of expensive FM in herbivorous species because of complementary digestive enzyme profile and the presence of ω-6 fatty acids from Azolla diet (Mosha 2018). In addition, fish fed Azolla supplemented diet resulted in improved protein conversion, mobilization and utilization of glycogenic amino acids. However, excess Azolla supplementation reduced fish growth and conversion efficiency, possibly due to the presence of high fiber content and low protein digestibility (Mosha 2018). O. niloticus and T. mozambicuss exhibited better growth performance in a range of 20–42% of dietary Azolla inclusion (Fiogbe et al. 2004, Ebrahim et al. 2007). Besides these, some reports propose positive growth even at 50% inclusion of Azolla meal (Almazan et al. 1986; El-Sayeed 1992). Nevertheless, despite being a microphagous omnivore fish, poor growth performance was reported in Tilapia zillii fed Azolla meal (Abdel-Halim et al. 1998). Similarly, O. niloticus and T. rendalli exhibited reduced growth pattern when fed Azolla incorporated diets (Micha et al. 1988). Literature suggests that various grades of Azolla levels have been incorporated in species belonging to family Cyprinidae. Improved feed utilization and growth rate was reported in rohu fed 10–50% Azolla meal in the diet (Tuladhar 2003; Datta 2011; Panigrahi 2014). While Orange fin labeo (Gangadhar et al. 2017), Catla (Umalatha 2018), silver carp and mrigal (Tuladhar 2003) and grass carp (Majhi et al. 2006) reported to have a range between 10–25% Azolla inclusion level in the diet (Kumari 2017). Our results from the current investigation are in line with the findings reported on above fish species with the inclusion levels of Azolla meal up to 10% that resulted in best growth performance of the candidate fish species.
Carcass composition is altered by numerous endogeneous and exogenous factors that often indicate quality of cultured fish species (Khan and Khan 2020a,b). Both protein and ash are controlled endogenously while as lipid values are altered by both of these factors. It is widely acknowledged that carcass protein and fat are the main attributes of interest and contribute to the suitability of fish meat for processing and storage. Further it has been suggested that feeding nutrient deficient diets results in impaired protein accretion and surplus fat deposition in liver, fillet or peritoneal cavity. In the present study, body protein decreased with higher inclusion of fishmeal with Azolla meal. This could be due to poor quality of plant protein compared to that of fish meal. However, the diets containing higher level of Azolla weed inclusion resulted in significantly high carcass moisture and lower lipid content. The high moisture and low lipid content of C. carpio communis fingerlings fed Azolla incorporated diets may be credited to the elevated plant protein derived from aquatic weed. Similarly, tilapia and common carp fed plant protein diets resulted in improved carcass moisture and reduced lipid content (Hassan and Edwards 1992; Hossain and Jauncey 1989). Datta (2011) reported that incorporation of Azolla resulted in reduction of body lipid content but their body protein content remained unaffected among all the treatments. Micha et al. (1988) reported the similar trend of body protein and lipid content in O. niloticus and T. rendalli fed Azolla supplemented diets in both the species.
One of the effective tools for understanding the physiological and pathological changes in fishes is ample knowledge of blood parameters. The blood parameters offer information on health status of a fish like metabolic disorders, deficiencies and chronic stress in response to changes related to diet, quality of water and ill health (Denson et al. 2003; Banaee et al. 2008; Khan and Khan 2021a,b,c,). Hematological parameters such as Hb, Hct%, RBCs showed an insignificant relationship with increase in dietary Azolla meal up to 10% (D2) beyond which significant decline was apparent. The highest values of these data were observed in D1 followed by D2 group. It is well known fact that Hb, Hct and RBC count are related to the non-specific immune function, where high Hb and RBC count can be taken as an indication of good health. Earlier studies on fish fed diet rich in plant protein sources exhibited reduced blood Hct and Hb (Pham et al. 2008; Lim and Lee 2008). It is a well-known fact that hematological parameters are drastically affected by imbalanced diets, presence of anti-nutritional factors and environmental conditions (Garrido et al. 1990; Lim and Lee, 2009). The results of current research work determined that a reduction in haematological values corresponds with the poor growth performance, especially in the D4 (30%) -D6 (50%) group.
Serum biochemical parameters are one of the useful indices for monitoring the health and physiological condition of fishes and are widely been used to determine the effects of feed additives on fish health (Fanouraki et al. 2007; Shi et al. 2006; Hoseinifar et al. 2010; Parrino et al. 2018; Fazio et al. 2019). These serum parameters can prove a pivotal tool for detecting illness and response to therapy. In aquaculture it is necessary to evaluate the serum biochemical parameters which enable us to know the normal physiological condition of the fish under study (Patrichi et al. 2011). Proteins are among the leading sources of energy in fishes and play a significant role in the blood glucose level in fishes (Shweta et al. 2012). In our study dietary inclusion of Azolla meal decreased total serum protein of C. carpio communis fingerlings which may be due to reduced rate of protein synthesis in fish fed higher levels of dietary Azolla meal. Potassium, phosphorous, calcium etc, are some of the commonly analyzed blood electrolytes. Calcium is a component of bones and also regulates nerve and muscle functions in fish. Potassium ion is majorly found in intercellular fluid and possesses an important function of carbohydrate metabolism in nerve fibers of animals including fish. Alteration in potassium concentration affects heart function and causes neurotoxic damage to the central nervous system of the fish (Adediji 2010). Potassium level in our study shows significant variation. Similarly significant variation was also found in calcium and phosphorous activity of fingerling C. carpio communis when compared with the group of fish fed basal diet (D0). On the other hand cholesterol shows the reverse trend means it starts to increase on higher inclusion of dietary Azolla. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are enzymes found in the different tissues of liver, kidneys, heart, skeletal muscle, pancreas, spleen, erythrocyte, brain and gills (Hadi et al. 2009). These blood plasma enzymes are calculated clinically as biomarkers for liver health. On injury these enzymes are being released directly into blood hence provide information about liver dysfunction. In our study serum ALT and AST increased significantly as FM was replaced with Azolla meal. It indicates that Azolla replacement of FM had negative impact on the liver. However, Xu et al. (2018) found that serum ALT and cholesterol levels were significantly lower than that of control group in C. carpio fed FM replaced diets. Serum glucose reflected the status of normal metabolism in the body and the healthy degree of liver function (Zhao 2006; Xu et al. 2018). Serum glucose levels were also found to increase with higher inclusion levels of dietary Azolla among all the groups. Our results suggested that fish was not able to maintain normal glucose metabolism at higher inclusion levels and causing burden to the liver metabolism which was also supported by the higher values of AST and ALT levels.