Dietary supplementation of wheatgrass powder to assess somatic response of juvenile grass carp (Ctenopharyngodon idella)

Consequence of dietary fish meal substitution with wheatgrass was evaluated by observing growth response, associated feed cost and survival of grass carp (Ctenopharyngodon idella) fingerlings for sixty days. Sprouted wheatgrass (Triticum aestivum) was prepared for its inexpensively rich nutrients. Four isonitrogenous test diets were formulated and applied as treatments (T) in triplicates (R). In the control (T1), basal inclusion rate of fish meal was 30%, of which 10% was replaced with wheatgrass powder in T2 and in T3 replacement was 20%. In contrast, 30% of basal fishmeal was replaced in T4. Grass carp fingerlings (6.38±0.21 cm and 2.83±0.36 g) were stocked in twelve aquaria (60×40×45 cm3) each containing 75 L water, at 10 fish per aquarium, fed test diets at 5% of body weight twice daily. Prominent effect of wheatgrass supplementation was found on food conversion ratio (FCR) and survival rates. The significantly lowest FCR was observed in T3 (2.13±0.42) followed by T2 (2.89±0.99), T1 (3.01±1.53) and T4 (3.05±0.94). Besides, fish survival rate was significantly improved in T2 (90%), T3 (93.33%) and T4 (93.33%) compared to the lowest survival in T1 (83.33%). In conformity, fish tolerance (LT50) to low pH stressor was also increased with wheatgrass supplementation. The other growth parameters among the treatments were statistically similar with highest specific growth rate and fish production in T3 (1.13±0.12 %/day and 2.28±0.13 tons/ha). Dietary wheatgrass did not affect the fish carcass composition rather gave better result to some extents. The significantly highest carcass protein and lowest moisture was retained in T3 (14.13±0.05% and 74.91±0.25% respectively), whereas comparatively higher lipid and mineral (ash) content was found in T1 (7.69±0.02% and 2.35±0.27% respectively). Importantly, feed formulation cost was reduced by 2.61, 4.89, and 7.71% in T2, T3, and T4 respectively compared to T1. Therefore, wheatgrass could be promising in juvenile grass carp diet.


Introduction
Searching alternative protein sources from non conventional feedstuff to sustain the aqua-feed industry has become a vital trend of research in the aquaculture world. The current reliance on fishmeal or fish oil for feeding cultured fish has already caused an enormous loss to the wild fisheries resources (Tacon and Metian, 2008;FAO, 2012). Besides, the increased demand, limited supply at soaring price as well as greater propensity of fish meal to pollute the environment have made it urgent to replace with less expensive protein sources to improve the sustainability of aquaculture (Martinez-Llorens et al., 2009). In order to satisfy the quest for finding competitive replacement of fish meal, scientists have switched over a number of dietary ingredients of both animal and plant origin (Booth and Sheppard, 1984;Goda et al., 2007;Audu et al., 2010). However, in most of the cases high cost of animal sources or antinutritional factors of some plant origin had limited their wider use in aqua-feed (Davis, 2015). Although, plant based fish diets always had financial advancement to offer at comparatively lower price because of their availability (Azeredo et al., 2017;Francis et al., 2001). Therefore, in

Stocking of Fish
The fry of grass carp (initial size: 6.38±0.21 cm and 2.83±0.36 g) was collected from local fish hatchery and stocked at a rate of 10 fish per aquarium. During fry transportation oxygenated plastic bags were used to avoid stress and injury. Thereafter the experimental fish were subjected to conditioning into glass aquaria at room temperature ranging 25-30°C for a period of 8-10 days at the beginning of the experiment. Throughout the conditioning period, fish were fed control diet twice daily (10:00 am and 4:00 pm) at approximately 3% of live body weight/day.

Wheatgrass powder preparation
Firstly, 3 clean plastic buckets were taken containing 1 kg of locally collected wheat seeds each for sprouting. Then the collected wheat seeds were washed and soaked in water for overnight. In the following morning the soaked seeds were sieved, wrapped with cotton cloth and kept in a perforated bucket covered with cloths for 24 hours. Subsequently, the germinated wheat seeds were spread over each (60x30x8 cm 3 ) of nine previously prepared (washed and sun dried) sprouting beds (trays). Water was then sprayed over the trays and covered it for 2 days. Thereafter the sprouted young yellowish wheatgrass were uncovered and brought to sunlight. Harvesting was done, when the seedlings became 6-7 inch long with dark green colors within 8 days, with the cut off stems and weighed. Prior to harvest, water was continued to spray over the sprouting bed twice daily (morning and evening). After harvesting, blanching was done where the green wheatgrass stems were boiled (over 100 ºC) for 7 minutes and immediately cooled down in a big bowl by adding ice and one pinch of sea salt (NaCl). As a thumb rule, blanching is done by boiling objects at 75 to 105ºC temperature for 1to 10minutes, in order to retard enzymatic activity, removing gases, setting color, improving texture, stopping the changes of flavor and leaching of water soluble sugars. The blanched wheatgrass ( Figure 1) were kept aerated in room temperature and then dried into a dryer to give it a crunchy texture. These were then crumbled with scissor and finally blended to produce fine powder to be used as fish feed ingredients ( Figure 1).

Ingredients selection and fish feed formulation
The feeding trial was supported with four different experimental diets that were formulated form the following ingredients based on their availability, nutrient profiles and market price ( Table 1). The test diets were formulated with a view to reducing (0, 10, 20, and 30%) dietary inclusion of fish meal (basal inclusion of 30% in control T 1 ) by adding wheatgrass powder in a progressive manner. Accordingly, the control diet (T 1 ) contained 30% fishmeal but no wheatgrass powder. Whereas in T 2 , 10% of fish meal was replaced with wheatgrass powder; hence it contained 27% fish meal and 3% wheatgrass powder. In order to represent 20% replacement, T 3 contained 24% fishmeal and 6% of wheatgrass powder. In T 4 30% of fishmeal was replaced with 9% inclusion of wheatgrass powder (Table 2). Pearson square method was followed to calculate the dietary inclusion rate of different ingredients in order to formulate isonitrogenous (around 31%) test diets. Sinking dry pellet feed (0.5 mm diameter) was prepared with extruded feed pellet machine and sun dried. Prepared feed were stored in air tight polythene bags at 4°C in refrigerator before feeding the fish. The proximate composition of the test diets was also determined (AOAC, 1990) that has been shown in Table 3. Some precautionary actions were also taken in preparing the ingredients for feed formulation such as measured mustard oil cake was soaked overnight and soybean meal was pre-boiled to minimize their glucocyanate effects.

Fish feeding trial, sampling and data analysis
Throughout the feeding trial, experimental fish were fed (hand feeding) with the respective test diets twice daily (10 am and 5 pm) at 5% of their body weight in each aquarium. Siphoning was employed to drain 25% of the aquarium water daily that ensured removal of uneaten feed and faces. However, same amount of clean water was also added daily to maintain the water level in the aquarium. Besides daily water exchange, each aquarium was completely drained fortnightly (during sampling) to keep environmental homogeneity. Sampling of fish and water quality parameters were done biweekly. Fish were sampled randomly from individual treatment to observe their average length and weight as well as their response to the test diets by calculating the growth parameters such as length gain (cm), weight gain (g), percent weight gain, specific growth rate (SGR, %/day), food conversion ratio (FCR), survival rate (%) and fish production (kg/ha). Fish carcass profile was also determined following the standard procedure of AOAC (1990). Furthermore, water quality parameters such as dissolved oxygen (mg/L), water temperature (°C), pH, ammonia and nitrite contents were measured using portable DO meter, thermometer, pH meter and ammonia testing kits respectively. Moreover, collected data were loaded in the computer and after final harvest, data were subjected to one-way ANOVA for statistical analysis (Snedecor and Cochran, 1994). The least significant difference was used for comparison of the mean values ascertained from different treatments by Duncan's New Multiple Range Test (Duncan, 1955).

Low pH stress test
Tolerance to low pH stressor (pH 3) of the experimental fish was also evaluated to determine the impact of dietary wheatgrass incorporation on the fish fitness. Therefore, after the completion of feeding trail, 6 fish from each treatment were randomly selected and transferred to a 20 L container holding water having pH 3. In order to prepare this low pH water (pH 3), deep tube-well water was strongly aerated for 24 h and gradually mixed with nitric acid (HNO 3 ). The aquaria for stress test were facilitated with continuous aeration and kept under ambient temperature. Time required attaining 50% mortality of the test fish was calculated as median lethal time (LT 50 ).

Production of sprouted wheatgrass
The total collection of wheatgrass powder from nine sprouting beds (trays) was 500g. In order to obtain this, each tray (60x30x8 cm 3 ) was initially sprayed with 250 g of raw wheat seeds. Notably, around 20% wheatgrass powder was retained from the sprouted live wheatgrass. Therefore, average sprouted wheatgrass production rate was 277.78 g (live weight) per tray.

Somatic response of grass carp to test diets 3.2.1. Growth parameters
Dietary substitution of any unconventional ingredients always raises the question about its palatability to the target fish. In this experiment, progressive inclusion of wheatgrass powder in the test diets were well accepted by the grass carp fingerlings as they fed steadily but actively and there was almost no feed left over after twenty minutes of feed delivery. Grass carp are herbivorous in nature but also readily accept formulated pellets under culture conditions (Ni and Wang, 1999;George, 1982). Therefore, having plant origin might have aided wheatgrass contributing to the palatability of the diets. However, wheatgrass powder has also been evaluated in the diet of catfish without hampering diets' acceptability (Nath et al., 2014;Islam et al., 2017). In case of initial length and weight there was no significant difference (P>0.05) among the treatments. After 60 days of grass carp nursing, the highest mean length gain (cm) was observed in T 3 (1.05±0.39 cm), followed by T 4 (0.97±0.28 cm), T 2 (0.95±0.29 cm) and T 1 (0.78±0.81 cm), although the values were statistically similar. In contrast, fish in T 3 attained maximum mean weight gain (g) of 2.68±0.18 g, but the lowest was found in T 4 (2.13±0.56 g). However, the differences were insignificant (P>0.05) among the treatments (Table 4). The short rearing period (60 days) and/or some experimental error could be responsible for statistical non-significance. In a similar fashion, the highest Specific growth rate (SGR, %/day) and fish production (tons/ha) were experienced in T 3 (1.13±0.12%/day and 2.28±0.13 tons/ha) and the lowest in T 4 (0.95±0.19 %/day and 2.04±0.24 tons/ha) without any significant difference (P>0.05). The observed SGR (%/day) values have outweighed the findings of Islam et al. (2017), who reported SGR values ranged from 0.46 to 0.77 %/day for stinging catfish (Heteropneustes fossilis) and that of Nath et al. (2014), who documented SGR value 0.29 %/day for Asian catfish (Clarias batrachus) in wheatgrass based feeding trials. Species variation and environmental factors might be the reason behind these differences. Again, the fish production in this experiment was continued to increase with the increase of dietary wheatgrass supplementation, till 20% of fish meal replacement with wheatgrass powder in T 3 , but dropped when the replacement rose to 30% in T 4 . Correspondingly, review of literature has made it known that substitution of fish meal with plant based ingredients up to a certain level in fish diets results in positive response but higher dietary substitution causes a reduction in growth and immune responses (Lin andLuo 2011, Mokrani et al., 2020).

Survival rate
Considering the survival rate, fish fed wheatgrass powder based diets showed significantly improved survival compared to control diet (T 1 ). Therefore, juveniles in T 3 and T 4 enjoyed the highest survival rate (93.33%) followed by T 2 (90%) which were statistically different (P<0.05) to the lowest survival in T 1 (83.33%) ( Table 4). The fabulous nutritional profile of wheatgrass, loaded with quality minerals (K, Ca, Fe, Mg, Na and S) and vitamins (A, B, C and E) besides the basic nutrients, had presumably contributed to the better survival with increasing supplement in the test diets (Mujoriya and Bodla, 2011;Anwar et al., 2015;Devi et al., 2015). Therefore, the overall findings re-emphasize that fishmeal substitution with non-conventional sources (animal or plant origin) to a certain level could be feasible in fish diet without limiting growth (Ayoola, 2010;Rana et al., 2015;Sing et al., 2016;Islam et al., 2017;Daniel, 2018;Osho et al., 2019;Rana et al., 2020).

Food conversion ratio (FCR) and feed cost
Food conversion ratio (FCR) is an effective parameter to evaluate a fish feed as it determines the required amount of feed for per unit of somatic growth. In this study, the lowest FCR was 2.13±0.42 found in T 3 that was significantly lower (P<0.05) than the highest FCR in T 4 (3.05±0.94). Although, the FCR value of T 2 (2.89±0.99) was statistically similar to other treatments (Table 4). Similarly, Rana et al., 2020; substituted fishmeal with jute leaf powder in the diet of mrigal (Cirrhinus cirrhosus) fingerlings and reported FCR values ranged between 2.81 and 3.30. Importantly, feed preparation cost was decreased with the increase of dietary wheatgrass powder in the test diets. For instance, the expense in formulating T 1 diet was the highest (49.85 BDT/Kg), which was subsequently reduced by 2.61%, 4.89%, and 7.71% in T 2 (48.55 BDT/Kg), T 3 (47.41 BDT/Kg) and T 4 (46.01 BDT/Kg) respectively (Figure 2). Thus the experiment signifies that replacement of fish meal with low cost wheatgrass powder in the diet of juvenile grass carp could substantially reduce feed cost and show conformity with the findings of Rana et al., 2020. Moreover, reduction in feed cost, worth more than 60% of total aquaculture cost (Gadzama and Ndudim, 2019), has brought wheatgrass powder under the shades of light as a promising ingredients in the diet of Indian major carps.

Fish carcass composition
Carcass composition of the tested fish has been shown in Table 5. The findings reveal that, significantly highest carcass protein and lowest moisture was retained in T 3 (14.13±0.05% and 74.91±0.25% respectively). In contrast, comparatively higher lipid and mineral (ash) content was found in T 1 (7.69±0.02% and 2.35±0.27% respectively) which were statistically similar with other treatments except the lowest lipid content in T 4 (6.68±0.27%). Besides, fiber and carbohydrate profiles did not vary significantly among the treatments. Notably, fiber content was increased with progressive addition of wheatgrass in the test diets. However, the observed protein contents are slightly lower but lipid and mineral (ash) contents are higher than the findings of Ashraf et al., 2011;who reported 74.30±0.07% moisture, 20.00±0.15% protein, 2.52±0.01% lipid and 1.4±0.2% ash content in grass carp. This variation might be due to the diet and age variation of the fish. To sum up, addition of wheatgrass in place of fishmeal did not affect the quality of fish greatly rather improved consumer digestibility to some extent by increasing carcass fiber. Nandeesha et al., 1995; also concluded that plant based diets had contributed to boost up carcass protein and fat levels in Indian major carps.

Water quality parameters
The water quality parameters (Table 6) of the trial aquaria ranged between, pH (7.82 and 8.84), dissolved (DO) oxygen (6.57 and 7.93 ppm), temperature (28 and 29.5 ˚C), ammonia (0.08 and 0.33 mg/L) and nitrite (0.07 and 0.66 mg/L) among the treatments throughout the study period. Consequently, the parameters were within the acceptable range for grass carp as well as fish culture (Swingle, 1967;Ni and Wang, 1999). Therefore, it could be summarize that the rearing water was safe for fish wellbeing and had no decisive effect on the response of fish to the test diets.

Low pH stress test
Adaptation to stressed condition is an important reference of fish robustness. In this experiment, after the feeding trials fish were subjected to low pH stress test (pH 3.0). From the literature, it is understood that pH below 4.0 is lethal to fish, whereas the recommended range is 6.8-9.0 (Swingle, 1967). Results showed that 50% of the fish in T 1 died sooner after 6 minutes (LT 50 = 6 minutes) of exposure to low pH stress than fish in other treatments. In contrast, the values of median lethal time (LT 50 ) for the fish in T 2 , T 3 and T 4 were 10, 14 and 16 minutes respectively (Figure 3). These imply that dietary addition of wheatgrass (high mineral content) had improved resilience of the test fish which conforms with the findings of Rana et al., 2020.  (Duncan's New Multiple Range Test). * significant at P≤ 0.05; ** significant at P≤ 0.01; NS non-significant at P˃ 0.05