Replacement of Dietary Crude Protein of Soybean Meal by Tobacco Leaf Protein Concentrate on Growth, Haematological and Biochemical Responses of Catfish, Clarias

: This study assessed the effects of replacing dietary crude protein of soybean meal with tobacco leaf protein concentrate (TLPC) on growth, haematological and biochemical parameters of catfish, Clarias gariepinus juveniles. The catfish (n=126, mean weight = 31.77±0.07g) were arranged in a completely randomized design with 6 treatments and 3 replicates with 7fish per experimental unit and fed for 40days. The treatments were 0, 20, 40, 60, 80 and 100% replacement levels of soybean meal with Tobacco leaf protein concentrate respectively. Diets had negative effects (p > 0.05) on mean weight gain (MWG), specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio (PER) across treatments. Fish in the control group recorded highest values for MWG (51.20±0.25g), SGR (2.40±0.01) and PER (2.48±0.02). Similarly, the best value for FCR (1.03±0.01) was recorded amongst the control group of fish. With the exceptions of WBC, NEUT, LYPH and MCV, significant differences ( p <0.05) were recorded in the values of Packed cell volume, red blood cells, Haemoglobin, PLT, MCHC and MCH across diets. The biochemical parameters recorded no significant differences ( p > 0.05) across treatments with the exceptions of CHO, HDL and LDL. Also, significant differences (p < 0.05) were observed in the values of GSH, SOD, CAT and MDA across treatments. The poor growth performance coupled with reduced physiological activities of TLPC at different inclusion levels, confirmed that it could not favourably replace soybean meal however, TLPC enhanced the enzymatic antioxidant activities in the experimental fish, consequently improving the health status of C. gariepinus.

The faming of aquatic organisms which accounts for over 50 percent of global fish production, is the fastestgrowing food-producing sector across the world. Globally, about 424 aquatic species are farmed, benefiting millions of people through the provision of food security, poverty reduction and sustainable livelihood (Galappaththi et al., 2020). Over the past two decades, aquaculture has developed tremendously to become an economically significant industry all over the world. Comparatively, the industry continues to grow at an average global annual growth level of 8.8% per year over all other animal food production industries (Onada and Ogunola, 2017). The sustainable expansion of aquaculture production basically depends on the availability of inexpensive quality feed, since fish feed constitutes approximately 60 -70% of the total production. Thus, further development of the aqua-feed industries requires a consistent supply of nutritive raw materials for feed formulation (Fagbenro and Adebayo, 2005).  The conventional groundnut and soybean meals are over-competed by man and his animals making the cost of producing animal feed overwhelming. Hence, to lessen the cost of feed in fish production it is imperative to replace the aforementioned convectional sources of protein with suitable alternatives (Hung et al., 2007;Monebi and Ugwumba, 2013). Considerable interest has been shown on the use of conventional plant oilseed meals such as groundnut, cotton seed, soybean and rapeseed (Agbede, 2005;Toko et al., 2008;Liu et al., 2011). In furtherance of the above search, the inclusion of plant-based ingredients in fish feed has increased markedly over the years (Krogdahl et al., 2010). This include the use of leaves from legumes as valuable feed source in aquaculture, because of their abundance and are also rich in protein and minerals (Edelman and Colt, 2016). In addition, oil-bearing seeds and oil cakes which are by-products of the vegetable oil industry are similarly sources of plant protein (Gatlin et al., 2007). They are high in protein and low in carbohydrate. However, their high fibre and cellulose contents limit their nutritional value in the feed of monogastric animals (Ghaly et al., 2012). Accordingly, to address the aforesaid challenges, leaf protein concentrate (LPC) is being used as source of protein when compounding animal feed, because it is potentially the cheapest, and most abundant source of available protein (Sodamode et al., 2013;Adeyemi and Osubor, 2016). The LPC is made by mechanically separating indigestible fibre and soluble anti-nutrients from much of the protein, minerals and vitamins in some fresh green plant leaves (Adeyemi and Osubor, 2016). Tobacco (Nicotiana tabacum L.), an herbaceous plant grown widely for cigarette production, has been investigated extensively in the past decades as a source of high-quality leaf protein. Studies revealed that 1 g of dried tobacco leaf contained 94-146 mg protein before the flue-curing process (Fantozzi and Sensidoni, 1983;Salvucci and Anderson, 1987). Besides, the ability of tobaccos to grow in high density enabled them to yield fourfold more protein per acre compared with soybeans (Sheen and Sheen, 1985;Lo et al., 2010). Also, studies have shown that tobacco leaves are rich in soluble plant protein, which has high nutritional and medicinal value (Vansuyt et al., 2003;Teng and Wang, 2012). The African mud catfish, Clarias gariepinus is a significant aquaculture fish across the world, and Nigeria remains the leading producer of this fish (Kaleem and Bio Singou Sabi, 2021). This species has been extensively cultured because of its easy breeding, rapid growth and considerable economic value . Hence, it is of utmost importance to identify appropriate alternative feed ingredient to replace soybean meal in the diet of catfish for its sustainable production. This aim of this study was to assess the effects of replacing dietary crude protein of soybean meal with tobacco leaf protein concentrate on nutrient utilization, growth, haematological and biochemical parameters of catfish, Clarias gariepinus juvenile.

MATERIALS AND METHODS
The experimental design: The experiment was a complete randomized design with six experimental diets in triplicates carried out for a period of 40 days, at the Fish Nutrition Unit of the Department of Marine Sciences, Faculty of Science, University of Lagos, Nigeria.
Preparation of Tobacco leaf protein concentrate (TLPC): 100g leaf from each of the freshly sampled weed was washed and pounded which ruptured the plant cell wall. The residue was squeezed to release leaf juice which contained protein. The separated leaf juice was heated at 80-90 °C for 10 minutes to coagulate the leaf protein. A rubber hose was used to siphon the coagulated protein from the whey, the protein coagulum was further filtered through muslin cloth and screw-pressed to remove the entire whey. The leaf protein was washed with water, repressed and sundried to get the leaf protein concentrate (Fellow, 1987;Agbede and Aletor, 2004;Agbede, 2005).
Experimental diet: Six iso-nitrogenous diets were formulated ( Table 1). The Nicotiana tabacum leaf protein concentrate was used to substitute soybean meal at graded levels in the diets. The Control diet (0% TLPC), diet 1 (20% TLPC), diet 2 (40% TLPC), diet 3 (60% TLPC) diet 4 (80% TLPC), and diet 5 (100% TLPC). Soybean meal, fishmeal and groundnut cake were used as protein source, while maize, starch and oil were used as energy source. All feed stuffs were milled, weighed and mixed thoroughly for even distribution of the feed ingredients. Water was added to aid starch gelatinisation and the dough was passed pelletizer and passed through a 2 mm die. The pelleted feed was air-dried, sealed in zip lock bags and stored until it's needed. The proximate composition of formulated diets is shown in Table 2.
Experimental conditions and feeding regime: Three hundred C. gariepinus juveniles were obtained from Iceberg farm, Ikotun, Lagos State, and were transported in a 25L gallon to the fish nutrition unit, Department of Marine sciences, University of Lagos. The fish was acclimatized in an 800L capacity canvas tank fitted to a flow-through system and fed with 2 mm commercial feed (Blue crown) for a period of 14days. A total of 126 fish with an average initial weight of 31.77±0.067g each were randomly allocated in eighteen plastic tanks (52.5 ×33.5 ×21.0 cm 3 ) at 7 fish per tank in triplicates per experimental group. The fish were fed thrice daily (8:0 0am, 12:0 0pm and 5:0 0pm) to apparent satiation with the experimental diets for 40 days. Water was changed every other day with dechlorinated water from a borehole to maintain good water quality. The dissolved oxygen ranged from 4.5 to 6.0 mg/L and temperature ranged from 28.0 °C to 29.0 °C throughout the experimental period. The fish of each tank were bulk-weighed once every 10 days to determine the average weight gain while the quantity of the feed fed for each period was also recorded.  Growth and Nutrient Utilization study: At the end of the feeding trial, the fish growth performance and Nutrient utilization indices were calculated following Bekhan et al. (2006): T2-T1 (day) x 100

4)
Protein Efficiency Ratio (PER) PER = Mean Weight Gain (g) Protein Intake (g) Where Protein Intake (PI) = Total feed intake x Protein content of feed.
Collection of blood samples for hematological and biochemical analysis: At the end of feeding trial, the fish were starved for 24hrs prior to sampling. Blood was collected from two fish per tank (n = 6 fish per experimental group) via the caudal vein using 2-ml syringe and 23-gauge needles. It was emptied into Heparin bottles for haematology and plain bottles for biochemical analyses.
Biochemical analysis: The protein (PRO), glucose (GLU), cholesterol (CHO), triglyceride (TG), high density lipoprotein cholesterol (HDL), and lowdensity lipoprotein (LDL) were analysed at the Department of Clinical chemistry laboratory, University of Lagos Teaching Hospital using an automatic biochemical analyser by the colorimetric method (Coz-Rakovac et al., 2008).

Determination of Serum antioxidant parameters:
The catalase (CAT), malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) were carried out in the Department of Clinical Chemistry laboratory, Lagos University Teaching Hospital.  Data analysis: Results are presented with means ±SD of three replicates. All data were subjected to one-way analysis of variance (ANOVA) performed using SPSS version 16.0 and followed by Duncan's multiple range tests. P -value of < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION
The results of growth and nutrient utilization parameters of C. gariepinus juveniles fed graded levels of Tobacco leaf protein concentrate (TLPC) are shown in Table 3. Dietary inclusion of TLPC significantly decreased (p > 0.05) weight gain, SGR, PER and increased FCR of C. gariepinus when compared with those fed the basal diet. The highest (51.20±0.25g) and lowest (11.50±0.27g) mean weight gains were recorded in groups of fish fed control diet and diet 5 respectively. Similarly, the best (1.03±0.01g) and worst (2.65±0.03g) values for FCR were recorded in groups of fish fed control diet and diet 5 respectively. In the present study, we were able to replace up to 100% soybean meal with TLPC. Soybean meal (SBM) is an alternative protein ingredient currently of great interest to the aquaculture industry. However, the presence of antinutritional factors and its high-cost limits its use in crude and processed forms in aquafeeds (NRC, 1993;Ogbonna et al., 2014). The results from this study showed that the experimental diets were poorly utilised by C. gariepinus juveniles as reflected in the poor growth performance compared with the control diet. This finding supports the claim by Adewolu and Adamson (2011), who reported reduced fish growth rate after feeding Amaranthus spinosus leaf meal as potential dietary protein source in the practical diets for C. gariepinus. Similarly, Adebayo (2017) observed that despite the high crude protein level in corn gluten meal and other plant protein materials, the formulated diets were poorly utilised by the Hetero-clarias Juveniles, which was reflected in their growth performance. Furthermore, poor feed intake was observed among the groups of fish fed with the experimental diets compared with the control diet. This also contributed to the repressed growth observed among the experimental fish (Adebayo and Aladejare, 2015). Additionally, the reduced growth performance of fish fed diets with TLPC may be related to the limiting level of methionine and leucine, high antinutritional factors which depressed the feed intake and growth in fish at high levels of plant protein (Ngugia et al., 2017). Contrary to the above findings, Fagbenro et al. (2017) observed, that the mean weight gain and percentage weight gain of C. gariepinus fed with test diets containing cassava leaf protein concentrate variety up to 40% substitution of Soybean meal were similar to those fed with the control diet. Similarly, Chavez et al. (2016) included water hyacinth leaf protein concentrate substituting up to 75% of soybean meal for white shrimp, Litopenarus vannamei, and recorded beneficial effects at 25% on growth compared to a control diet.  The results of WBC, PCV, Hb, RBC, neutrophil, lymphocyte, MCHC, MCH and MCV counts of experimental diets are shown in Table 4. WBC, neutrophil, lymphocyte and MCV counts showed no significant differences (p > 0.05) between the control and other experimental groups fed graded levels of TLPC. Also, significant differences (p < 0.05) were recorded in the values of PCV, Hb and RBC counts, with the fish group fed with control diets recorded the highest values across treatments. Similarly, MCHC and MCH counts differed significantly (p < 0.05) between the control and other experimental groups of fish fed graded levels of TLPC diets. The results of haematological variables in this study suggest that the test diets did not precipitate any severe effects on the health status of the experimental fish because all the hematological parameters measured in this study were within the recommended physiological ranges reported for Clarias gariepinus (Fagbenro et. al., 2013;Eyiwunmi et al., 2018). Because, red blood corpuscle, white blood corpuscle, neutrophils, and lymphocytes are indicators for fish health feed and anti-nutritional toxicity (Ozovehe, 2013). Moreover, differences in values of haemoglobin (Hb), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular haemoglobin (MCH), blood platelets, and packed cell volume (PCV) between the fish of control group and others, indicated the healthy status of experimental fish (Abdul Kari et al., 2021).
The results of TLPC on serum protein (PRO), CHOL, TG, HDL, LDL, GLU, ALB and GLO of C. gariepinus are shown in Table 5. The values of serum protein, triglyceride, albumin, globulin and glucose recorded no significant difference (p > 0.05) between the fish of control group and others. However, slight differences were recorded in the values of CHO, HDL and LDL of fish fed with the control diet and diets 3 to 5. Albumin, globulins and total proteins ensure a healthy system and function as plasma carriers (Nya and Austin, 2009). In this study, the aforementioned parameters did not differ significantly across all groups, indicating healthy status of all the experimental fish. These results were in congruence with Kumar et al. (2010), who observed no significant effect in the protein and globulin levels in C. carpio fed 50 and 75% detoxified Jatropha curcas kernel meal. Lipids are biomolecules used by fish to stock energy and can be stored in many different organs (Guijarro et al., 2003). The transport of lipids and other lipid-soluble components from the intestine to peripheral tissues is predominantly mediated by lipoproteins (Botham and Mayes, 2016). The ratio of HDL to LDL is vital because high levels of HDL and low levels of LDL as recorded in this study, are needed to reduce the chances of coronary heart disease (Ali et al., 2012). Therefore, the values of CHO, TG, LDL and HDL recorded with the test diets, N. tabacum leaf protein concentrate showed no adverse effect on the state of health of experimental fish.  (Kurutas, 2016;Zhang et al., 2019). SOD is one of the major antioxidant defense enzymes produced in response to oxidative damage, which converts toxic superoxide anions into hydrogen peroxide (Wang et al., 2018), while Catalase is an enzyme found in nearly all living organisms exposed to oxygen, including bacteria, and animals which catalyzes the decomposition of hydrogen peroxide to water and oxygen (Chelikani et al.,2005). The elevation of CAT, GSH and SOD activities, and the reduction in the values of MDA in this study, suggest an adaptive response related to oxidative damage in order to counteract the impact of increased reactive oxygen species (ROS) generation. These results were in agreement with Zhang et al. (2022) who reported that dietary Flos populi extract fed to gibel carp (Carassius auratus) significantly increased serum superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) activities, while the content of malondialdehyde (MDA) in serum decreased significantly. Similarly, dietary supplementation of Moringa oleifera to sodium fluoride exposed fish (Nile Tilapia, Oreochromis niloticus) resulted in a significant reduction in MDA levels, and a significant elevation of SOD, CAT and GSH activities in a timedependent manner (Ahmed et al., 2020). Additionally, Aluta et al. (2021) affirmed that Quantitative RT-PCR showed that SOD expression in hepatic tissues was upregulated as a result of feeding C. gariepinus with onion supplemented diet. Conclusion: The poor growth performance recorded in this study indicated that tobacco leaf protein concentrate, could not favourably replace soybean meal however, the dietary TLPC enhanced the enzymatic antioxidant activities in the experimental fish, consequently improving the health status of C. gariepinus.