Study site and work design: The current work was accomplished at the Fish Seed Hatchery, Faisalabad, Pakistan, and three-month feeding trial was conducted in glass aquaria for the period of 90 days. GIFT fingerlings were obtained from fish seed hatchery Faisalabad that were hatched from selectively breed.

Experimental design: A total of 135 apparently healthy Genetically Improved Farmed Tilapia (GIFT) fingerlings of mean initial length 8 cm and weight 30 g were acclimatized for 15 days in rectangular glass aquaria measuring (40 × 30 × 40 cm, 96 L capacity containing 90 L of water) with the experimental conditions. Physicochemical parameters (pH, Temperature, DO, Hardness, Alkalinity etc.) of water were monitors in optimum range throughout the experimental duration.

Diets preparation and feeding regimes: The commercially available feed consisting of rice bran, wheat bran, grains, cereal products, vitamins, and trace minerals was used as a basal diet in the present study. Whereas blood was collected from the main slaughterhouse in Faisalabad for diet preparation. The experimental diets were formulated by mixing fish meal with blood meal and soya bean meal as animal and plant protein, respectively. Feed pellets were packed and stored at 4 °C in clean-dry plastic containers until use. The chemical composition of the diet was tested by using standard methods [9] before the experimental diet formulation. All ingredients were ground into a fine powder and then mixed well in a twin shaft paddle mixer until a solid dough was obtained. Subsequently, the dough was passed through a lab extruder (SYSLG30-IV Experimental Extruder) along with the addition of water [10]. The diet was dried for 2 h in a ventilated oven at 60 °C and mixed with oil slowly. Diet ingredients and proximate composition are shown in Table I. Fish grouping: Fingerlings were randomly allocated into three groups and kept in 9 aquariums. The first group (control group) was fed on a basal diet with 35% crude protein.  The second group (plant protein) was fed on a diet mixed with 35% plant protein. The third group was (animal protein) fed a diet supplemented with 35% animal protein over a period of 90 days.

Fish and tissues sampling: Fish and tissue sampling was performed at the end of the experiment (3-4 from each). Randomly selected fish were euthanized using 0.02% benzocaine [11] and their body length-weight was taken in centimeters and grams respectively. Fish dorsal muscles were frozen in liquid nitrogen and the samples were later on stored at -20°C until evaluated for their proximate composition.

Study of Growth: By using a standard formula, the growth parameters such that, specific growth rate (SGR), weight gain (g) and weight gain percentage of fish were evaluated.

Weight gain % = (Final weight – Initial weight) × 100
                                        Initial weight

SGR% = (ln. final wt. of fish – ln. initial wt. of fish) × 100
                                          Test day

Hematological analysis: Fish were starved for 24 h before blood sampling at the end of the experimental period. Fish from each group were randomly selected and blood samples were collected, under anesthesia, from the caudal vein using buffered tricaine methane sulphonate (MS-222) (100 μg/mL). A blood sample was divided into two parts to determine hematological and biochemical indices along with antioxidant activities. The first was transferred into a sterile tube containing 0.5% ethylene diamine tetra acetate (EDTA) (EDT001, Bioshop, Canada) and used to estimate white blood cells (WBCs) according to the method of [12] respectively. Furthermore, red blood cells (RBCs) were determined using the standardized cyanomethemoglobin procedure. The other portion of the blood sample was transferred into a sterile centrifuge tube, clotted, and centrifuged at 4000 rpm for 15 min at 4 °C for serum separation. The separated serum was stored at −20 °C until required for estimation of biochemical and antioxidant activities.

Statistical analysis: Differences between treatment groups were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s test. Differences were considered significant at p < 0.05. All results are expressed as the mean± standard error. All statistical analyses were performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA).

Results

The water pH and temperature, ranged from 6.90 to 9.00 and 25.0 °C to 32.0 °C respectively. Dissolved oxygen, total hardness and alkalinity ranged from 4.0 to 8.0 mg/L, 980 to 1930 mg/L, and 200 to 290 mg/L, respectively. The total dissolved solvents (TDS) and salinity concentrations ranged from 4 to 8.5 mg/L, and 1.00-3.00 ppt, respectively as given by [13]. The conductivity of water varied between 32.00 μS/cm and 89.00 μS/cm in different aquariums.

Growth parameters and somatic indices: The results of growth performance of GIFT fed on different diets are presented in Table III. There was no significant differences in the mean initial body weight (32.89 g) of GIFT among all experimental groups. The final body weight, weight gain, average body weight and specific growth rate (SGR) were significantly varied in fish groups fed on animal protein and plant proteins supplemented diets in comparison with the basal diet throughout the study period. The final body weight, weight gain and specific growth rate (SGR) were 52. 62 ± 0.74 g, 19.84 ± 1.56 g, 62.67 ± 7.06 %, 0.22 ±0.02 g and 0.53±0.05 (% g/day) respectively in group I, 70.36 ± 0.93 g, 37.58±1.95 g, 118.25±10.97 %, 0.42±0.02 g and 0.86 ± 0.06 (% g/day), respectively in group II. In case of animal diet group III, these parameters were found with values of 97.59±1.36 g, 64.48±1.22 g, 197.82±10.69 %, 0.72±0.01 g and 1.21±0.04 (% g/day), respectively. The highest values for growth performance indices were recorded in the animal supplemented group (p<0.05). There was no significant increase in total length and but significant in standard length of GIFT among all feeding groups on 90th day of sampling.

The proximate composition of GIFT fed on basal diet and experimental diets are illustrated in Table VI. After three months of feeding trial, crude protein, lipids, moisture, total ash and crude fibers were recorded 11.13±0.39, 1.30±0.09, 74.83±0.19, 0.76±0.05 and 0.66±0.04, respectively in group I. The crude protein (40.90±0.25) and lipid (2.57±0.07) contents of fish fed animal protein containing diet were significantly higher (p= 0.000) compared to 32.36±1.40 and 1.91±0.04 values of fish fed on plant protein containing diet. The moisture content showed no significant differences among all groups. Total ash (1.77±0.05) and crude fiber (1.11±0.04) contents were significantly increased in plant protein-based diet group compared to the control group (p=0.000), while these contents (ash=1.47±0.01, crude fiber=0.92±0.01) were lower in animal protein diet group when compared to fish fed on plant protein containing diet (p = 0.000). The quality of crude protein, lipid, total ash and fiber varied among fish kept on different diets. The amount of crude protein and lipids was highest in fish kept on animal protein diet followed by plant protein diet and basal diet. On the other hand, crude fiber and total ash content were highest in fish kept on plant protein followed by plant protein.

Hematology and serum biochemical profile including blood protein profile, liver function enzymes and immunity of GIFT fed on experimental diets in comparison with the control group are presented in Table V. The serum ALT, AST and ALP activities were found increased in diet supplemented with plant protein. These levels were higher than those in fish fed basal and animal protein supplemented diets. There was a significant elevation of serum albumin and globulin levels in experimental diets when compared with the basal diet group. Meanwhile, no significant differences were observed in WBCs among all experimental groups when compared with the control. The RBC count was significantly higher (p < 0.05) in the diet supplemented with animal protein than the other groups.

Figures & Tables

The aqua feed normally contains 25 to 50% protein content, which is principal and expensive ingredient in feeds and is essential for growth and maintenance of animal health [14]. Fishmeal is the most common protein source used in aquaculture feeds which account for 68% of global fishmeal consumption. It contains high levels of nucleotides, minerals, vitamins, fatty acids, balanced amino acid profile, high protein palatability and digestibility [15]. The shortage in world production of fishmeal makes it necessary to search for high- protein feed resources.

Micronutrients, which include minerals and vitamins, are required in lower quantity and play essential role in cellular functions. The dietary requirements in terms of composition and quantity of different nutrients can vary considerably among and within species. One of the most important external factors that influence fish feeding behavior and growth is food. These mechanisms are influenced by both food availability and dietary composition. Growth of animal is a polygenic and environmentally (e.g., nutrition) controlled phenotypic expression of muscle hyperplasia [16].

The obtained results clearly revealed that replacing up to 35 % of the fishmeal with animal protein enhance growth parameters including final weight final body weight, weight gain, and average body weight of GIFT when compared to those of control group. Increased growth performance can be attributed to high protein and essential amino acid profiles in blood meal that makes it perfect replacement of fishmeal in fish diet [17]. Blood meal can totally substitute fishmeal with no deleterious effects on growth, survival and feed conversion of Nile tilapia and catfish. The nutrient digestibility values of fishmeal and blood meal are significantly similar for most tilapia species [18] implying that blood meal can be used as a fishmeal substitute in the diet. The fish fed 35% experimental diet of fermented blood meal and unfermented blood meal exhibited significantly higher growth performance than fish fed a control diet of 100% fishmeal [19]. In the current study, although 35% plant protein inclusion level improved the growth performance as compared to control diets. However, growth rate was lower than those fed on animal protein diet. The results suggest that protein and energy digestibility for plant protein is apparently lower than that of animal protein for fish. Possible reasons for the reduced feed utilization and growth parameters recorded for plant protein-based diet may be related to the presence of high-crude fiber, anti-nutritional factors, and poor palatability.

Measures of size and proximate composition of juvenile hatchery-reared fish are necessary to determine if different diets lead to changes in growth or nutrient provisioning, and ultimately survival. The effect of diet on proximate composition in hatchery-reared fish has been determined in a variety of species and it remains one of the most simple and effective means of evaluating dietary quality [20]. In this study, fish fed animal and plant protein-based diets had significantly higher whole-body crude protein and crude lipid content than the control group. However, fish fed the animal protein containing diet had highest whole-body moisture, protein, and lipid contents than those fed the plant supplemented diet. These results are consistent with [21], they found that a high plant protein-based diet reduces protein utilization, carcass protein content, protein retention, and growth.

The hemato-biochemical analysis is an important parameter to determine physiological performance of animal since it can reveal information about hematopoiesis, liver, kidney, and immune system. As a result, hemato-biochemical indices are valuable to detect metabolic disorders, alterations of the functions of various organs and health monitoring [22]. Liver has a complicated structure and plays an important role in the preservation, implementation, and regulation of the body homeostasis [23]. High levels of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) are the crucial markers to detect liver injury. Similarly, serum level of alkaline phosphatase (ALP) is also associated with status and function of hepatic cells since synthesis of this enzyme increases due to rising biliary pressure [24].  In this study, ALT and AST levels were recorded to be higher with inclusion of animal protein in diet. However, ALP levels did not differ significantly amongst the experimental diets. In a similar study, a positive linear relationship was found between incremental amount of animal protein (housefly maggot meal) and serum AST and ALP levels [25]. Fish fed a dietary inclusion of plant protein had higher AST and ALT activity than fish fed a control diet, which is consistent with a study in which significantly higher ALT and AST levels in serum were found in juvenile seabass fed diets supplemented with commercial soya bean (above 30%) as plant protein source The findings indicate that liver of GIFT fish may have been damaged to some extent, which in turn affected the protein metabolism.

Since the liver synthesizes serum proteins, including albumin and a small fraction of globulin, these proteins suffer both quantitative and qualitative alterations in any hepatic disease. The concentration of serum albumin and globulin falls in any disease that causes hepatocellular injury. Serum albumin and globulin levels change over time, providing useful indicators of hepatic disease severity, progression, and prognosis. GIFT fed on animal protein supplemented diet had considerably higher serum albumin and globulin protein levels compared to fish fed on plant protein supplemented and control diets. These findings suggest that a diet supplemented with animal protein improves fish health. The differential analysis of WBC counts is valuable tool in measuring physiological status and helpful in the evaluation of immunity status of fish [26]. Blood profile is generally linked with physical condition as its values are excellent signals of dietary condition [27]. In the present study, mean RBCs count of GIFT significantly increased with inclusion of 35 % animal protein-based diet compared to the fish fed the control diet. In another study, [28] evaluated the effects of replacing fishmeal with animal protein source on hematological and biochemical parameters in common carp (C. Carpio) fingerlings. The authors found that fish fed experimental diets for 14 weeks have improvement in hematological parameters (RBC, WBC, Hb and HCT) and plasma proteins (albumin and globulin) in comparison with control group. In a similar study the effects of replacing fishmeal with different percentages of plant protein (dietary fermented soy pulp) to assess growth performance, hematology, and blood biochemical of African catfish and found the significant increase in RBC and lymphocytosis in fish fed the protein diet. The albumin and globulin were significantly higher (p < 0.05) in the experimental diets compared with the control diet [29].

GIFT has the capacity to survive a wide range of adverse climatic conditions. It could be one of the most favorable specie for production in Pakistan. The present study concluded that dietary supplementation with animal protein in the form of cattle blood had positively influence the growth performance, proximate composition, and hematological parameters of GIFT. Expensive fish meal may be replaced with cheap animal blood from slaughterhouse as animal protein source in aqua feed for optimal culturing of tilapia fish.

Author Contributions

Hafiza Samra Ambreen conducted trial for research, collected data and prepared manuscript 
Najma Arshad assisted in idea conception, Supervised, revision of manuscript critically for important intellectual content.
Muhammad Mudassar Shahzad design experiment, supervised throughout study trial and help in critical review of manuscript preparing
Ghulam Ayesha Javed contributed to analysis, interpretation of data and helped in manuscript preparing
Kiran Shahzadi made contributions in proof reading and review.

Conflict of Interest

The authors have declared no conflict of interests.

Acknowledgment

The authors would like to thank the Punjab Fisheries Department, Lahore for providing facilities for research.

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