The results obtained in the present study clearly indicate that the use of organic herbal spice seeds in fish diets have pronounced effect on the growth, haematological performance, digestive enzyme activity and stress parameters of the fish. In the present study, T8 (Coriander seed powder, 2%) showed the best growth performance throughout the experiment. At the end of the experiment, maximum length and weight was observed in T8 was 10.04 ± 0.34 cm and 11.48 ± 0.19 g respectively which was significantly higher than control (7.46 ± 0.42 cm and 8.53 ± 0.27g respectively). The results are in agreement with Al-Shakarchi and Mohammed (2021) who has fed coriander supplemented diet (1.75%) to Common carp (Cyprinus carpio) which resulted in enhanced growth performance as compared to the control group. Similar results have been reported by Raissy et al. (2021) who fed Common carp (Cyprinus carpio) with Coriander (Coriandrum sativum), Oak acorn (Quercus brantii) and Mallow (Malva sylvestris). T8 also showed the best results in terms of bio-growth parameters such as Weight Gain (8.103 ± 0.160g), Daily Weight Gain (4.00 ± 0.04%), Specific Growth Rate (2.04 ± 0.01%), Relative Growth Rate (L) (191.75 ± 5.86%), Relative Growth Rate (W) (240.34 ± 2.59%) etc. These results are in accordance with Farsani et al., (2019) who fed Rainbow trout (Onchorhynchus mykiss) with different levels of coriander supplemented diets and found out significantly higher values of Total weight gain, Daily weight gain and Specific Growth Rate (SGR) in the treatment group fed with 2% coriander seed incorporated diets. Enhanced growth performance in fish fed with coriander can be attributed to the effect of bactericidal property of coriander seeds on the intestinal flora which leads to better nutrient absorption and in turn higher metabolism and better feed utility responsible for enhanced growth performance (Citarasu T, 2010; Awad and Awaad, 2017; Begnami et al., 2018). Better growth prospects in fish fed with coriander seeds could be related to the presence of some specific biochemical compounds such as Terpenes and Coriandrons present in coriander seeds (Nadeem et al., 2013). It is evident that the spice or herbal seeds have appetite-inducing and digestion-stimulating properties that play an important role in feed utilization and conversion hence leading to better growth performance (Jain et al., 2008).
Haematological indices are a significant tool for monitoring fish health in general and response to nutrition in particular (Secombes 1996; Cuesta 2005; Lin et al., 2015). As per the results obtained from the present study, highest RBC count (1.46 ± 0.6×106/mm3) was recorded in T9 (Coriander seed powder, 2.5%) while as highest WBC count (27.70 ± 4.7×103/mm3) was observed in T8 (Coriander seed powder, 2.0%). These findings are in agreement with Farsani et al. (2019) who have fed Rainbow trout (Onchorhynchus mykiss) with different levels of coriander supplemented diets and found out the best haematological performance in diets supplemented with 2% coriander. T8 also showed the highest values of haemoglobin and haematocrit and followed an increasing trend throughout the experiment. Increase in blood cell counts can be attributed to the effect of bio-active compounds in coriander seed on the lymphoid organs of the fish (Ellis, 2001) which results in increased production of cells in the blood. RBC count not only indicates the health status of the fish but is a reliable indicator of stress in fish (Talpur and Ikhwanuddin, 2013). Any abnormality in RBC count in blood either indicates anaemia in fish or presence of stress (Talpur and Ikwanuddin, 2013). In the present study, the RBC counts of the fish fed with different treatment diets did not vary greatly from each other in terms of blood cell counts, only a slight variation among different treatment groups and between treatment groups and control was observed (Maximum value, T9 (1.46 ± 0.6×106/mm3), Minimum value, T3 (1.21 ± 0.7 ×106/mm3). Hence, it can be assured that as per the results obtained for blood indices, the fish in the present study did not undergo any stress. Similar results have been put forth by Raissy et al. (2021) who related the highest growth performance in coriander supplemented diet group to the effects of coriander essential oils (in coriander seeds) that have a stimulatory effect on the haematopoietic organs of the fish responsible for the production of RBCs and increase the oxygen carrying capacity of the cells. Total Protein, Albumin, and Globulin are considered the most important tools of monitoring the fish health prospect in terms of immunity (Ahmadi et al., 2012; Rao et al., 2006; Sahu et al., 2007 ). In the present study, Blood protein (Albumin and Globulin) was observed the highest (6.42 ± 0.1g/dL) in T7 (Coriander seed powder, 1.5%) in comparison to other treatment groups and control. Highest value of glucose (118.47 ± 12.09mg/dl) was recorded in control throughout the experiment. Lowest glucose level (92.47 ± 22.00mg/dl) was recorded in T9 (Coriander seed powder, 2.5%). Significant decrease in blood glucose levels in treatment group fed with 2.5% coriander seed powder supplementation may be due to the hypoglycemic property of coriander seeds (Laribi et al., 2015). Specifically, the hypoglycemic effect of coriander seeds on fish blood glucose can be better explained as a result of the combined effect of certain bioactive compounds present in coriander seeds such as Linalool, Geranyl Acetate and γ-terpinene (Gallagher et al., 2003; Pandeya, 2013).In certain fishes including Tilapia, wide distribution of different digestive enzymes like protease, trypsin, lipase and amylase in addition to intestinal length promotes the possibility of digesting a wide range of food sources and significantly improves the metabolic process of food breakdown into blood streams responsible for tissue formation and overall growth (Montoya-Mejia et al., 2017).
In fishes, the digestion of nutrients begins with the activity of digestive enzymes including Trypsin, Chymotrypsin, Amylase and Lipase secreted by the pancreas into the stomach and continues till the intestines (Moriarty, 1973; Nagase, 1964). In the present study, different treatment groups fed with different herb supplemented diets were subjected to digestive enzyme analysis (Trypsin, Lipase and Amylase). Trypsin activity is considered as one of the conditional indicators of fish, and its secretion is directly proportional to the activity of the pancreas (Sunde et al., 2001). In the present study, highest Trypsin activity (4.414 ± 0.5U/mg protein) in the gut was observed in T2 (Celery seed powder, 2%) and lowest (0.038 ± 0.4 U/mg protein) in T6 (Fenugreek seed powder, 2.5%). Enhanced Trypsin activity in Celery fed group is in line with the study carried out by Mohamed et al., (2018) who fed Common Carp (Cyprinus carpio) with celery supplemented diet in three different proportions and observed a consistent increase in the Trypsin levels with increase in Celery supplementation in diets. Trypsin levels in treatment fish fed with Fenugreek supplemented diets were observed the lowest compared to other treatment groups and control diet fed groups. Lowest trypsin levels may be due to the presence of characteristic Trypsin inhibitors present in Fenugreek seed (Al-Maiman, 2004; Mansour and El-Adawy, 1994; Oddepally et al., 2013; Pallavi and Rajender, 2021). Lipase enzyme is mainly secreted by the pancreas and plays a major role in lipid breakdown, especially tri-acyl-glycerols, necessary for better digestion (Awad, 2010). In the present research study, highest Lipase activity (0.932 ± 0.09 U/mg protein) was recorded in T6 (Fenugreek seed powder, 2.5%) while as the lowest Lipase activity (2.456 ± 0.1 U/mg protein) was found in control group. The result can be related to the possible explanation that among all the three herb ingredients used in the present study, Fenugreek constitutes the highest (6–7%) lipid content (USDA, 2012) for the breakdown of which, Lipase production in fish digestive tract shows a significant increase with the increase in supplementation levels (Mehboob et al., 2017) compared to other experiment ingredients (Celery and Coriander seed) with negligible lipid content. Similar results have been reported by Al-Saraji and Nasir (2013) who fed the Common Carp (Cyprinus carpio) with different protein source supplemented diets and observed an increase in Lipase levels in Fenugreek supplemented diet fed group compared to other treatment groups and control. Amylase enzyme production in fish is stimulated by the presence of glycogen, glycolytic chains, starch and higher dietary protein content in fish diet (Krogdahl et al., 2005). Maximum Amylase enzyme activity (0.932 ± 0.09 U/mg protein) was observed in T6 (Fenugreek seed powder, 2.5%) and minimum (0.124 ± 0.01 U/mg protein) in T1 (Celery seed powder, 1.5%). The results are evident by the fact that Fenugreek seeds are exceptionally high (23–26%) in protein content (USDA, 2012) in comparison to Celery and Coriander with meager dietary protein content. The results obtained are in line with the results reported by Kawai (1973) who found an increase in Amylase enzyme in the gut of Rainbow trout (O. mykiss) fed with Fenugreek as a source of dietary plant protein.
Oxidative stress is accompanied by an increase in the production of Reactive Oxygen Species (ROS). In the present study, highest values for oxidative enzymes were recorded in liver followed by gill and muscle. In the liver, highest Catalase activity was recorded in T9 (2.6630 ± 0.64 U/mg protein). In gill, highest activity was recorded in T9 (2.4187 ± 0.025 U/mg protein) and in muscle, highest Catalase activity was found in T9 (1.9150 ± 0.013 U/mg protein). Although, very scanty reports are available on the relationship between oxidative enzymes and herbs used in the present study, however, highest value of Catalase in T9 (Coriander seed powder, 2.5%) can be possibly related to the high content of catalase in celery (Kolarovic et al., 2009). Under stress, anaerobic glycolysis supplies energy accompanied by a significant accumulation of lactate (LDH) under hypoxic conditions (Wang et al., 2013). In the liver, highest LDH activity was recorded in T9 (16.8560 ± 0.192 U/mg protein). In gill, highest activity was recorded in T9 (14.039 ± 0.061 U/mg protein) and in muscle, highest LDH activity was found also found in T9 (13.7903 ± 0.020 U/mg protein). Zhao et al. (2020) reported that fish exposed to hypoxic and ammonia conditions exhibits higher lactate concentration and LDH activity, confirming the occurrence of anaerobic glycolysis under simultaneous exposure. In the liver, highest MDH activity was recorded in T9 (2.9407 ± 0.068 U/mg protein) followed by gill (2.4620 ± 0.077 U/mg protein), where as in muscle, highest MDH activity was found in T8 (1.9113 ± 0.140 U/mg protein). High stocking density can also cause oxidative stress in fish, because of the imbalance between reactive oxygen species and antioxidant defense systems (Mittler, 2002; Jia et al., 2016). In fish body these are removed by the antioxidant enzymes such as SOD, GPx, and Catalase (Birnie-Gauvin et al., 2017). In the liver, highest SOD activity was recorded in T9 (2.9820 ± 0.068 U/mg protein) followed by gill (2.2610 ± 0.032 U/mg protein) and muscle (2.0767 ± 0.068 U/mg protein). Wang et al. (2013) observed an increase in the activity of antioxidant enzymes in fish under high stocking density conditions, indicating that the adaptive response of the antioxidant system protects the body from oxygen free radicals during stress. In the liver, highest NBT activity was recorded in T9 (1.412 ± 0.126 U/mg protein) followed by gill (1.116 ± 0.159 U/mg protein) and muscle (0.879 ± 0.018 U/mg protein). Previous research studies have revealed that acute hypoxia induced oxidative stress has altered antioxidant enzyme activities in Largemouth Bass (Micropterus salmoides) (Yang et al., 2017). Similar results were also obtained for Spotted Croaker (Leiostomus xanthurus) exposed to hypoxia (Cooper et al., 2002).