Further Study on Toxicity of the Permisable (Very Low) Level of Ochratoxin A with and without Essential Oils on Rats

Published on: 3/11/2020
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An indoor feeding experiment for 57 days was conducted on Nile tilapia to evaluate the effects of dietary inclusion of graded levels (0, 25, 50, 75, and 100%) of sieving wastes meal of Egyptian clover seeds instead of soybean meal based on crude protein content. Glass aquaria (each of 70x35x40cm, filled with 65 l dechlorinated tap water, stocked with 12 fingerlings 17.3 g initial body weight and 9.5cm total length) were used. The obtained results recommended the best replacement level of 50% which realized significantly the best feed composition, highest feed consumption, final body weight, and best fish body composition, to be reflected in many benefits for both the producers and the consumers.


Nowadays aquaculture in Egypt supplies ca. 80% of human fish consumption. Egypt occupies the tenth rank all over the world concerning aquaculture production (FAO, 2016). But the Aqua-feeds are shorter than the demands, besides their expensive costs; therefore there is a need to recycle and reuse all possible and available field and industrial by-products that have a nutritional importance for fish (Koeleman, 2018a & b). Since fish are the most feed converter than all terrestrial animal species (Shehab, 2018). However, adult tilapia is herbivores, omnivores, or detritovores (El-Sayed, 1991).
So, many authors evaluated fish diets containing various agro-industrial wastes (El-Sayed, 1999) as fish meal, soybean meal, linseed meal, corn gluten, tomato waste, clover leaves, duck weed, poultry by-product meal, and bone and meat meal (Eid et al., 1995), sugar beet pulp (Magouz, 1996), lupine seed meal (Hassanen, 1998), corn gluten meal (El-Ebiary et al., 2001), black seed and roquette seed meals (Abd Elmonem et al., 2002), sunflower meal (El-Saidy and Gaber, 2002a & c), dried dropping dates (Srour et al., 2002), sugarcane bagasse compost (Hafez et al., 2003), sesame hulls by-product (Abd Elmonem et al., 2004), fennel seed meal, dried marjoram leaves, and dried basil leaves (El-Dakar et al., 2004a, b, & c, respectively), leaf protein concentrate from water hyacinth (El-Saidy et al., 2004), olive cake (Khalafalla and Salem, 2006), and silkworm meal (Salem et al., 2008), etc. Therefore, the present paper aimed to study the possibility of feeding Nile tilapia diets containing sieving wastes meal of Egyptian clover seeds instead of soybean meal.
Materials and Methods
Mono-sex Nile tilapia fingerlings (17.3g initial body weight and 9.5cm total length) were purchased from a private hatchery at Tolombat 7, Kafr El-Sheikh governorate. After transportation to Sakha Aquaculture Research Unit, Central lab. of Aquaculture Research, Abbasa, Abou Hammad, Sharqia governorate, Agricultural Research Center, Cairo; fish were adapted on the experimental lab. conditions for two weeks in a plastic tank of 1000 l capacity. Then distributed into 65 l glass aquaria, each of 70 x 35 x 40 cm stocked with 12 fingerlings, two aquaria/treatment. Aquaria were provided with dechlorinated tap water, air compressors, air stones and heaters with thermostats. Aquaria water was partially replaced with syphoning five times weekly and completely once a week. Rearing water criteria were measured periodically. Natural light was available 10 h daily. Five iso-nitrogenus (25.36 – 25.92% crude protein) diets were formulated to contain graded levels of sieving wastes meal of the Egyptian clover seeds instead of the dietary soybean meal on basis of crude protein content. Table 1 illustrates the experimental diets' formulation. Diets 1, 2, 3, 4, and 5 contained 25.92, 25.78, 25.64, 25.36 and 25.36% calculated crude protein.
Table 1: Formulation of the experimental diets (Kg)
All dietary ingredients were purchased from the local market; where, the gluten (62% crude protein) was purchased from Badamas Feed Factory belonging to Dakahlia Company for Poultry and rice bran (4000, 12.5% crude protein) from the Rice Blower at Al-Gamal country, Mansoura. Corn (imported from Argentine) contained 8% crude protein was purchased from Dakahlia Company for Poultry at Badamas then was ground on 3 mm sieve at Al-Ezz factory of feeds. Broiler concentrate (Holman) was purchased from Dakahlia Company for Poultry at Badamas. It consists of mineral salts and vitamins as given in Table 2. Diets were offered five days a week at daily feeding rate of 3% of the actual body weight of fish, at two meals daily 9 am and 12 mid-day. Fish were weighed bi-weekly to readjust feed quantity. Diets and whole fish body were chemically analyzed using AOAC (2000) procedures. Growth performance [body weight, total length, specific growth rate, relative growth rate, condition factor (Froese, 2006), and mortality rate] and feed utilization (feed intake and feed conversion ratio) were measured or calculated. Numerical data collected were statistically analyzed using SAS (2006) and Duncan (1955).
Table 2: Specifications and ingredients of Holman broiler concentrate 45% protein (Alpha Feed International, Al Obour – Industrial Zone) Reg. No. 8813
Results and Discussion

Some water quality criteria: The rearing water criteria periodically measured from 19/11/2017 to 16/1/2018 did not differ among treatments or throughout the experimental period. Since water temperature ranged between 28.20 and 28.65ºC, pH 7.85-8.70, dissolved oxygen (DO) 9.10-9.85 mg/l, total ammonia (T.amm.) 0.70-0.85 mg/l, and salinity (Sal.) 1.20-1.55‰ (Table 3). These conditions are suitable for rearing Nile tilapia (Abd El-Hakim et al., 2002).
Table 3: Mean values of some quality criteria of the fish rearing water throughout the experimental period 
Proximate analysis of the experimental diets: The gradual levels of the replacement (sieving wastes meal of the Egyptian clover seeds instead of soybean meal in the fish diets) significantly and gradually increased each of the dietary contents of dry matter, crude protein and carbohydrates; but significantly and gradually reduced the dietary contents of both ether extract and crude fibers (Table 4, because of the high mortality rate in the fish group fed the diet of 25% replacement, it was excluded). These results are in partial agreement with those of Abdelhamid and Saleh (2015) and Abdelhamid et al. (2016). It was proved that the increase of dietary CP increases significantly the body weight during certain physiological phases, and affects significantly and positively the RGR as well as significantly influences each of feed intake and dry matter content of fish body (Abdelhamid et al., 2001 and Khalil et al., 2001).
Table 4: Mean values of the chemical analysis for the experimental diets, % dry matter basis  
Feed consumption of the experimental fish: Feed consumption by the experimental fish differed significantly by the dietary replacement levels. The highest feed intake (P≤0.01) was calculated for the fish group fed at 50% replacement comparing with the other replacement levels (Table 5). These results are confirmed by those of Abo-Donia et al. (2004), Abdelhamid and Saleh (2015) and Abdelhamid et al. (2016) with some terrestrial animals.
Table 5: Means and standard errors of feed intake throughout the experimental period (g/fish/57 days) 
Final live body weight of the experimental fish: Concerning body weight of the experimental fish, there were no significant (P>0.05) differences among dietary treatments at the start of the experiment; yet, there were significant variations among treatments at the end of the experiment. Where the final body weight of the treatment group fed with 50% sieving wastes meal of Egyptian clover seeds instead of soybean meal was significantly (P≤0.01) heavier than those of the other treatment groups (Table 6). This improvement in the final body weight of that treatment may be due to the significant increase in feed consumption of those group's fish (Table 5), whereas the lower final body weight of fish fed the two highest replacement levels (75 and 100%) may be related to the significant high content of carbohydrates and crude fibers and significant low fat contents of their diets (Table 4). These results are in line with those of Abo-Donia et al. (2004), Abdelhamid and Saleh (2015) and Abdelhamid et al. (2016) although the variation among species of the experimental animals. In the same direction, replacement of 25% freshwater crab meal or 50% mixer (1/1) of duck weed meal with freshwater crab meal instead of fish meal in Nile tilapia diet led to the best final body weight, body weight gain, daily body weight gain, feed conversion rate, and economic efficiency as well as the highest CP and the lowest EE of fish body and flesh) Abdelhamid et al., 2009a & b; 2011 and 2012b).
In another research field for feed evaluation of unconventional carbohydrate resources (one of agro-industrial by-products, i.e corn wastes of "Karata" manufacture) instead of corn in tilapia diets, the 100% replacement rate significantly succeeded; since, it was responsible for the best final body weight, body weight gain, daily body weight gain, RGR, SGR, feed intake, feed utilization, and fish body CP content (Abdelhamid et al., 2011). Recently in the field of recycling the agro-industrial by-products in fish feeding, Abdelhamid et al. (2015) proved that the DDGS could be used in tilapia diets at 20% replacement level instead of fish meal to obtain the best growth performance; whereas, 100% replacement level led to the best economic efficiency. Moreover, Khadr (2018) found that 30% replacement rate of the DDGS did not affect negatively fish performance and health, but even improved the economic efficiency.
Table 6: Means and standard errors of final body weight (g/fish) of the experimental fish
Growth rates of the experimental fish: Table 7 illustrates the response of the experimental fish to the dietary treatments tested in form of their growth rates. Throughout the experimental period, both of relative growth rate (RGR) and specific growth rate (SGR) negatively affected by all replacement levels, so reflected lower values than the control (0% replacement level). In an evaluation for plant protein sources, Abdelhamid et al. (2004a) reported that mallow (Malva parviflora L.) plants meal could replace 10% of the dietary protein as an alternative protein source for Nile tilapia (Oreochromis niloticus) fingerlings without negative effects on fish growth performance, fish muscular area, and economic efficiency. In another trial, Abdelhamid et al. (2004b) found that it was possible to replace 10% soybean meal protein by sesame meal protein in Nile tilapia fingerlings diet without any negative influences on growth performance, feed and nutrients utilization, and fish body composition. Moreover, it could partially (25-50%) and successfully replaces soybean meal protein by black seeds (Nigella sativa) meal protein in Nile tilapia diet, which realized the best growth rates and economic efficiency when black seeds meal is available (Abdelhamid et al., 2005b).
Water hyacinth protein could also partially (10-20%) replace soybean meal protein realizing the best growth rates and economic efficiency (Abdelhamid et al., 2006). Yet, Abdelhamid et al. (2010a & b) confirmed opposite results; since the dietary inclusion of water hyacinth (particularly from polluted resources) negatively affected fish weight, their SGR, survival, feed conversion, and fish body composition. So, they recommended to does not use water hyacinth from polluted water resources in fish nutrition, but when it is necessary and in case of feed shortage, it could be used only from uncontaminated resources and a maximum limit not exceed 30% of the dietary soybean meal protein. Although many people in Kafr El-Sheikh Governorate (as the highest governorate allover Egypt in producing fish) talk about sewage using in fish nutrition; yet, different freshwater fish species (Nile tilapia, silver carb, common carb, and African catfish) reflected low values of all tested measurements (growth, feed utilization, and fish body composition, dressing, and boneless meat) by their feeding with sewage comparing with those fed commercial-artificial diet. So, Abdelhamid et al. (2014) recommended do not feeding fish with treated sewage.   
Table 7: Means of RGR and SGR of the tested fish throughout the experimental period

Final total length and condition factor of the experimental fish: The calculated condition factor (CF) of the experimental fish at the start of the experiment was 2.018%, since the initial body weight and total length were 17.3 g and 9.5 cm, respectively. Values of final total length and condition factor of the experimental fish (Table 8) revealed no significant (P>0.05) differences among treatments; yet, fish fed the 50% replacement diet gave superior CF (2.33%) than all other treatments. This superiority of CF with that diet may be due to the significant highest final body weight of fish in that treatment (Table 6) than with all other treatment groups. Sieving wastes of berseem (clover) seeds were rarely used in animal nutrition (Abo-Donia et al., 2004; Abdelhamid and Saleh, 2015 and Abdelhamid et al., 2016) but there is no available data on its use in fish nutrition. Generally, in the field of using novel (unconventional) feeds, dried live yeast as a source of mono-cellular protein was used with/or without LactoSac in Nile tilapia diets. Its mixture (at a level of 20g/Kg diet) was the best in significant improving the weight, length, condition factor, and feed utilization of fish (Abdelhamid et al., 2000).

Table 8: Means of the final total length and condition factor of the experimental fish (each value is the mean of 8 fishes) 

Feed conversion ratio (FCR) of the experimental fish

Although the significant elevation of the feed consumption by fish in the treatment offered the 50%-replacement diet; yet, it reflected the best feed conversion ratio (0.90), since it gave the significant highest final body weight than all other treatments. The values of FCR were 1.47, 0.90, 2.68, and 2.30 for the diets included 0, 50, 75, and 100% replacement levels, respectively. Feed evaluation process continued, Abdelhamid et al. (2005a) successfully replaced till 50% of soybean meal protein by linseed meal protein in Nile tilapia diets and obtained improved growth, feed conversion, nutrients utilization, fish body protein content, and economic efficiency.

Mortality rate of the experimental fish: The mortality rate was equal (50%) in both treatments contained 0 and 75% replacement, but it increased somewhat (54.1%) at 100% replacement. Yet, it was relatively low (33.3%) with 50% replacement diet.

Chemical analysis of the whole fish body end of the experiment: Table 9 presents mean values of the chemical composition of the whole fish body at the end of the experimental period. There is a gradual and significant (P≤0.01) decrease in moisture percentage, i.e. increase in dry matter content in fish body by increasing the replacement level. This led to significant (P≤0.01) increase in crude protein (CP) and ash contents by the increase of replacement percentage. The opposite was true, concerning ether extract (EE) that decreased significantly (P≤0.01) with increasing the replacement rate. Hassanen et al. (1995) reported that increasing levels of some fermented waste foods (tomato pulp silage) in diets reduced lipid content but increased ash content in whole fish body as that found in the present work. Also, Kheir and Sweilum (1997) reported that increasing dietary CP content led to increase fish body CP and decrease its ash content. Since the negative relationship between CP and EE is a fact (El-Saidy and Gaber, 1997, 2002b & 2005; Hassanen, 1998; Azab et al., 2002; Abd Elmonem et al., 2002; Gaber, 2002a & b & 2006; Zaki and El-Ebiary, 2003; El-Dakar et al., 2004b; Gaber and Hanafy, 2004; Abdelhamid et al., 2006, 2009b, 2011, 2012b, & 2015; Diab et al., 2006; Hanafy, 2006; Soltan et al., 2006; Saad, 2007 & 2010; Ali, 2008; Salem et al., 2008 and Farrag et al., 2013).

Although other researchers reported positive correlation between fish body CP and EE (Kheir and Sweilum, 1997; Mabrouk et al., 2000; Soltan et al., 2001; El-Saidy and Gaber, 2002a & b; El-Sayed, 2003; Shalaby et al., 2003; El-Dakar et al., 2004a & b; El-Saidy and Gaber, 2004; Nour et al., 2004; Ayaad and Hassouna, 2005a & b; Gaber, 2005; Eweedah et al., 2006 and Soltan et al., 2008); but others (Mohamed and Hanafy, 2002; Soltan, 2002; Soltan et al., 2002 and El-Dakar, 2004) found no effect on fish chemical composition due to dietary treatments. However, carbohydrates did not influence significantly by the dietary treatments. The improvement in chemical composition of the whole fish body (concerning CP content) may be attributed to the increase of dietary CP content with increasing the replacement level (Table 4).

By evaluating medicinal plants in fish nutrition, Abdelhamid and Soliman (2012a &c) obtained significant improvement in Nile tilapia utilization of energy and protein of the diets included fenugreek seeds or Cresson seeds at 1-2% levels, so the growth rates and fish body protein increased. Also, Abdelhamid and Soliman (2012d) recommended the dietary inclusion of 2% dried leaves of guava or camphor trees for its improving effects on growth performance, feed utilization, and fish body composition. Additionally, Abdelhamid and Soliman (2013) recommended also the addition of 1-2% of the common carb diets either of Mentha arvensis leaves meal, Camellia sinesis leaves meal, Marticaria chmomilla meal, Origanum majorana leaves meal, Zingiber officinale meal, or Cinnamum zeylanicum meal (according to its availability and prices to preserve fish production economy) because its beneficial roles in improving growth performance, feed and nutrients utilization, and fish body composition. On the other hand, Magouz (1996) found that dietary inclusion of sugar beet pulp did not influence Nile tilapia body composition.

Table 9: Means of chemical analysis of whole body of the experimental fish at the end of the experiment (% fresh weight basis) 


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