Comparison of organic and inorganic microminerals in all plant diets for Nile tilapia Oreochromis niloticus

Accepted Manuscript Comparison of organic and inorganic microminerals in all plant diets for Nile tilapia Oreochromis niloticus

Lay Nguyen, Fernando Kubitza, Shimaa M.R. Salem, Terry R. Hanson, D. Allen Davis PII: DOI: Reference:

S0044-8486(18)30102-9 doi:10.1016/j.aquaculture.2018.08.034 AQUA 633476

To appear in:

aquaculture

Received date: Revised date: Accepted date:

30 January 2018 13 August 2018 15 August 2018

Please cite this article as: Lay Nguyen, Fernando Kubitza, Shimaa M.R. Salem, Terry R. Hanson, D. Allen Davis , Comparison of organic and inorganic microminerals in all plant diets for Nile tilapia Oreochromis niloticus. Aqua (2018), doi:10.1016/ j.aquaculture.2018.08.034

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ACCEPTED MANUSCRIPT Comparison of organic and inorganic microminerals in all plant diets for Nile tilapia Oreochromis niloticus

Lay Nguyena,* [email protected], Fernando Kubitzaa, Shimaa M. R. Salemb, Terry

School of Fisheries, Aquaculture and Aquatic Science, Auburn University, 203 Swingle Hall,

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R. Hansona, D. Allen Davisa

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Auburn, AL 36849, USA

Department of Nutrition and Nutritional Deficiency Diseases, Faculty of Veterinary Medicine

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Mansoura University, 35516 Egypt

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Corresponding author.

ACCEPTED MANUSCRIPT Abstract An 8-week feeding trial was conducted to evaluate the production performance of all male Nile Tilapia (Oreochromis niloticus) fed diets supplemented with increasing levels of trace mineral premixes (copper, selenium, zinc, iron, manganese) from inorganic (I) and organic (O)

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sources. A practical trace mineral basal diet was developed using only plant ingredients. Nine

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experimental diets were prepared from the basal formula by adding graded levels of inorganic or

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organic trace mineral premixes to deliver 0 (control diet), 0.5, 1.0, 2.0 or 4.0 times the micromineral requirement for tilapia as stated in the NRC (2011). Juvenile Nile tilapia (initial

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weight 7.13 ± 0.24 g) were randomly stocked into 50-L aquaria at 25 fish per aquarium. Each of

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the nine treatments was replicated five times except treatment 8 (I-4) with four replicates. According to the results of our study, there were no significant effects of trace mineral premix

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levels and sources on the growth performance, survival rate and whole body proximate

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composition of Nile tilapia (P > 0.05). However, the trace mineral concentrations in the whole body and fillet were significantly influenced by the dietary levels of premixes. With the

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exception of selenium, there were no major differences in the micromineral contents in the fillet

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between fish fed the organic or inorganic sources of microminerals. Fish fed diets with organic selenium (Selplex, Alltech®) had significantly higher selenium levels in the fillet with the

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adjusted mean of 0.0191 mg/100g fillet compared to 0.0161 mg/100g in fish fed inorganic selenium (Na-Selenite). Keywords: Nile tilapia, inorganic, organic, microminerals, growth performance.

ACCEPTED MANUSCRIPT 1. Introduction Aquaculture has achieved global significance, supplying half of the seafood and fisheries products consumed worldwide (FAO, 2016). Continued growth of sustainable aquaculture has demanded a shift from the use of marine proteins and oils toward the use of plant ingredients in

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aquaculture feeds. As plant feedstuffs are generally known as limited sources of certain minerals

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and often contain factors that reduce the mineral bioavailability, dietary trace minerals

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supplementation is commonly recommended to ensure the optimum growth and health of cultured species.

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Despite being required at very small amounts in the diet and body, trace minerals or

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microminerals are important components of hormones and enzymes, serve as cofactors and/or activators of a variety of enzymes and participate in a wide variety of biochemical processes

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(NRC, 2011). Among many microminerals, iron (Fe), manganese (Mn), copper (Cu), zinc (Zn)

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and selenium (Se) have been demonstrated as essential nutrients to be supplemented in the diet of many cultured fish due to their low levels in practical feedstuffs and/or interactions with other

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dietary components (Watanabe et al., 1997). The deficiencies of Cu (Davis et al., 1993), Mn

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(Lall, 2002), Zn (Lin et al., 2008), Se (Lee et al., 2016), Fe (Gatlin and Wilson, 1986) in feed induced poor growth rate and high mortality in many species of aquatic animals.

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Tilapia is the second largest group of fish cultured in the world, with nearly 5.6 million metric tons produced in 2015 (Fitzsimmon, 2016). Regardless of the advances in fish nutrition, the information on the trace mineral requirements for Nile tilapia, as well as for other fishes, is still limited and fragmentary (Eid and Ghonim, 1994; El-Serafy et al., 2007; Lee et al., 2016; Sa et al., 2004). This is mainly due to the complexities arising from the ability of aquatic animals to absorb minerals from the surrounding water and from natural food (algae, microcrustaceans,

ACCEPTED MANUSCRIPT worms and others) available in pond environment to satisfy part of their nutritional requirements, as well as from requirement variation in response to salt regulation or osmotic pressure. Traditionally, inorganic salts of trace mineral have been used in commercial aquatic feeds as supplements to improve the levels of microminerals in the diets and prevent mineral

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deficiencies of fish and shrimp. Since the cost of inorganic minerals is relatively low, limited

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research effort has been spent in this area with the exception of those minerals which have been

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set for the limits of discharge into aquatic environments (Davis, 2015). There is also a tendency to overdose inorganic minerals in commercial feeds to ensure a generous safety factor.

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Chatvijitkul (2015) conducted a study to evaluate the chemical composition and pollution

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potential of 203 manufactured fish and shrimp feeds. Results of this study showed that all the feeds were considerably higher in Cu, Fe, Mn, Se and Zn than the required levels reported by

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NRC (2011). The overdosing of trace minerals is a concern to natural populations of aquatic

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animals, and the excess of certain metals in animal tissues can also pose a health risk for humans who consume contaminated meat (Fernandes et al., 2008).

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Concurrently, chelation of minerals has been employed in animal feeds to enhance

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absorption of trace minerals (Ashmead, 1992). Compared to inorganic salts, minerals chelated to organic molecules (organic bound minerals) have been reported to have higher bioavailability in

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practical diets by preventing the strong absorption of trace elements into insoluble colloids (Buentello et al., 2009; Hardy and Shearer, 1985; Lin et al., 2013; Paripatananont and Lovell, 1995a, b, 1997; Scott et al., 1982; Shao et al., 2010). Structurally stable chelated minerals are less sensitive to the inhibitory action of phytate in practical, plant-based diets (Ashmead, 1992; Garcia-Aranda et al., 1983). There are several published reports on the potential benefits of using chelated premix (Cu, Zn and Mn) in rainbow trout (Apines et al., 2003; Apines-Amar et al.,

ACCEPTED MANUSCRIPT 2004) rockfish (Katya et al., 2016) and Pacific white shrimp Litopenaeus vannamei (Bharadwaj et al., 2014) showing better response of these aquatic animals with the supplements of organic elements over inorganic sources. Worth mentioning, Katya et al., (2016) suggested 2~4 times higher body saturation efficiency of organic chelated Cu and Zn over inorganic forms of these

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minerals by observing the trend in the Cu and Zn content of whole body of the marine rockfish

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Sebastes schlegeli.

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Given the expected benefits of organic trace minerals over inorganic in supporting growth of fish, the present study sought to evaluate the effects of different levels of traditional

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inorganic and commercial chelated (Alltech®) sources of trace minerals (Cu, Zn, Fe, Mn and Se)

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on the growth performance of Nile Tilapia.

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2. Material and methods

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2.1. Experimental diets

A basal diet was formulated using only plant ingredients to meet the nutritional

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requirements of Nile tilapia (36% crude protein, 6% lipid and 3,200 kcal/kg of digestible energy;

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Table 2). Graded levels of trace mineral premix derived from organic (O) or inorganic (I) sources (Table 1) were added to the basal diet to provide 0.5, 1.0, 2.0 or 4.0 times the NRC (2011)

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suggested micromineral requirements for tilapia (30 mg/kg Zn, 85 mg/kg Fe, 7 mg/kg Mn, 4 mg/kg Cu, 0.25mg/kg Se and 1 mg/kg). A non-supplemented basal diet was kept as a control. The mineral premixes and test diets were prepared at the Aquatic Animal Nutrition Laboratory at the School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University (Auburn, Alabama, USA). Pre-ground feed ingredients and soybean oil were mixed in a food mixer (Hobart Corporation, Troy, Ohio, USA) for 15 min. Hot water at a boiling point was added at

ACCEPTED MANUSCRIPT 30% to the complete mixture in order to attain an appropriate consistency for pelleting and improve starch gelatinization. Diets were then formed through a 1.8-mm diameter die in a meat grinder, air dried at < 50°C to a moisture less than 11% and stored in the freezer at -20°C until used. Samples of 150 g of each diet were sent to Midwest Laboratories (Omaha, NE, USA) for

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proximate and mineral analyses (Table 3) (AOAC 930.15, AOAC 990.03, AOAC 945.16,

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Ankom.Tech, AOAC 942.05 were used for moisture, protein, fat, fiber and ash analysis

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respectively; AOAC 985.01 was used for mineral analysis except selenium (USP <233>)). 2.2. Fish and experimental conditions

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2.2.1. Culture method

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The study was conducted at the E.W. Shell Fisheries Center, Auburn, Alabama. All-male, sex-reversed Nile Tilapia (Oreochromis niloticus) fingerlings produced at the center were used in

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the experiment. The sex reversal and the fingerling rearing phases were performed in indoor

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nursery tanks, in which fish were fed commercial diets. Eleven hundred fish were stocked into forty-four 55-L aquaria supplied with a continuous water flow and aeration (25 fish per

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aquarium). Fish were fed the control diet twice a day during one week of acclimation period.

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Any eventual dead fish were replaced during this period. At the end of the acclimation period, fish of each aquarium were group weighed (mean weight of 7.13 ± 0.24 g), counted and returned

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to the same aquarium for the start of the experimental period. During the study, fish were fed close to apparent satiation which the equal ratios for all treatments varied from 4 – 5% of body weight daily. Every two weeks all the fish in each aquarium were group weighed and counted to estimate total fish biomass and average weight of fish, allowing for the adjustment of feeding allowance. 2.1.2. Water quality monitoring

ACCEPTED MANUSCRIPT The fish were stocked in the semi-closer recirculating aquaculture system in which water replacement was performed at 15 – 20% per day. The aquaria were provided with continuous water flow rate of 0.45 L/min and aeration by diffused air. The forty-four 55-L aquaria shared a single 3.65 m3 recirculating system, with a sump, a fluidized bed biological filter and a bead

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filter. Water temperature was maintained at 26 to 28oC using a submerged 3600 W heater

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(Aquatic Eco-Systems Inc., Apopka, Florida, USA) and pH were kept at 6.5 to 7.5 through the

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addition of sodium bicarbonate as need. Dissolved oxygen (DO) and water temperature were measured twice per day using YSI 650 multi-parameter instrument (YSI, Yellow Springs, Ohio)

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while pH, total ammonia nitrogen (TAN) and nitrite-nitrogen were measured twice per week.

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Photoperiod was set at 14h light and 10h dark. During the experimental period, DO, temperature, salinity, pH, TAN, and nitrite were within acceptable ranges for tilapia at 5.99 ± 0.70 mg/L,

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26.99 ± 2.10°C, 2.03 ± 0.86 ppt, 7.29 ± 0.49, 0.10 ± 0.08 mg/L and 0.04 ± 0.02 mg/L,

2.2.3. Sampling and analysis

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respectively.

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At the end of the experiment fish of each aquarium were group weighed and counted to

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determine average weight and overall survival. Five fish from each aquarium were collected and frozen for subsequent analyses of whole body and fillet proximate and mineral composition.

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Proximate and mineral composition was analyzed by Midwest Laboratories (Omaha, NE, USA). The following performance and nutritional parameters were evaluated after the completion of the study: a) Thermal-unit growth coefficient (TGC) = (final weigh1/3 - initial weight1/3) / (temperature × day) × 100. b) Apparent net protein retention (ANPR, %) = (final weight × final protein content) -

ACCEPTED MANUSCRIPT (initial weight × initial protein content) × 100 / protein intake. c) Protein efficiency ratio (PER) = weight gain / protein intake. d) Whole body mineral retention = (final weight × final whole-body mineral – initial weight × initial whole-body mineral) × 100 / mineral intake.

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2. 3. Statistical analysis

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All data were subjected to a one-way and two-way analysis of variance to determine

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significant differences (P < 0.05) among the treatments, which was followed by Tukey’s multiple comparison test to distinguish significant differences among treatment means. The

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analysis of covariance ANCOVA was performed to compare the two regression lines of fillet

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micromineral concentrations of Nile tilapia response to the use of inorganic trace minerals versus organic trace minerals. All the data were analyzed using SAS (V9.4. SAS Institute, Cary, North

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Carolina, USA).

3.1. Growth performance

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3. Results

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Percent weight gain (433.63 - 454.54%), thermal-unit growth coefficient (0.0950 0.0975), feed conversion ratio (1.56 - 1.64) and survival of fish (96.8 - 100%) are summarized in

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Table 4. Apparent net protein retention ranged from 26.0 to 26.9% and protein efficiency ratio from 1.64 to 1.72 g of gain/g protein. No significant effects (P > 0.05) of sources and doses of microminerals were observed in all these parameters. 3.2. Proximate composition of whole body fish samples Whole body moisture (72.47 - 74.04%), crude protein (15.27% - 15.60%), lipids (6.57% 7.92%) and ash (3.48% - 3.67%) contents of tilapia fed the experimental diets are summarized in

ACCEPTED MANUSCRIPT Table 5. These variables were not influenced (P < 0.05) by the source or the level of trace minerals in the diets. There was also no significant effect of the interaction among sources and levels of microminerals on whole body proximate composition of fish. 3.3. Whole body trace mineral concentration

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Trace mineral concentrations in whole tilapia are presented in Table 6. Whole body Zn

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concentrations ranged from 23.58 to 26.15 ppm and were not significantly influenced by dietary

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source or level of minerals. However, the level of mineral supplementation significantly influenced the whole-body composition of Cu (2.62 - 3.77 ppm), Fe (14.42 - 21.64 ppm), Mn

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(1.28 - 2.03 ppm) and Se (0.18 - 0.53 ppm). Cu level in whole fish was only significantly higher

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in tilapia fed the diet I-4.0 compared to the control diet. Whole body Fe concentration was also higher (P > 0.05) in fish fed either O-4.0 or I-4.0 (21.61 ppm and 21.64 ppm, respectively),

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compared to the control diet (17.42 ppm). In contrast, whole body Mn levels decreased as dietary

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levels of this mineral increased. Fish fed either the control (2.00 ppm) or the I-0.5 diet had significantly higher levels of Mn compared to fish fed other experimental diets. Whole body Se

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levels increased as the dietary mineral premix inclusion increased (P < 0.05) and reached the

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highest concentration (0.53 ppm) in fish fed diet I-4.0. However, the source of Se did not influence the whole body Se concentration of fish fed diets supplemented at 0.5, 1.0 or 2.0 times

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the NRC Se levels.

3.4. Mineral retention

Mn was the least retained (0.9 - 2.6%), while Se showed the highest retention ratio (22 32%) among all the microminerals supplemented (Table 7). Except for the Se, the whole-body retention of other trace minerals (Fe, Cu, Mn and Zn) decreased with the increase of mineral

ACCEPTED MANUSCRIPT premix inclusion in the diets (P < 0.05). There were also no major differences in the retention of microminerals between the organic and inorganic sources (P > 0.05). 3.5. Mineral contents in the fillet The concentration of trace elements in the fillet of tilapia (expressed as mg/100 g) of raw

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fillet ranged from 0.045 to 0.068 for Cu, 0.46 to 0.544 for Fe, 0.005 to 0.016 for Mn, and 0.628

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to 0.676 for Zn, and were not influenced by either the source or the dietary concentration of these

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trace minerals (Table 8). In contrast, the concentration of Se in the fillet increased with the increase in the dietary levels of Se either in fish fed diets supplemented with the organic or the

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inorganic mineral premix. Supplementation with organic Se (Se yeast) resulted in higher average

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Se concentration, 0.0197 mg/100g (P < 0.05), compared to 0.0161 mg/100 g of fillet using diets

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with inorganic source (Na-Selenite).

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4. Discussion

The quantification of proper mineral’s supplementation in practical diets for fish is

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difficult as fish can absorb some minerals from the water, a fraction of the minerals may leach

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from the experimental diets, feed ingredients contain a certain amount of minerals. In addition, the interaction of some minerals among themselves or with vitamins and other dietary

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components (e.g. phytate) can influence their bioavailability, making availability unpredictable. Some species of fish, particularly tilapia, may obtain significant amounts of minerals from natural food such as microalgae and microcrustaceans. Limited research has been conducted to estimate the trace mineral requirements of Nile tilapia Oreochromis niloticus, since tilapia have been traditionally cultured in pond environment in the presence of abundant natural food, making mineral deficiencies less unlikely to happen and hard to be assessed. However, with the

ACCEPTED MANUSCRIPT intensification of tilapia culture in the last decades using high density cages, raceways and recirculating systems, more research on mineral requirements of tilapia are needed. Nile Tilapia requirements for Zn (Eid and Ghonim, 1994; Sa et al, 2004) and Fe (Barros, 2002; El-sefary, 2007) have been evaluated using purified and practical diets while the requirements of Mn (Lin

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et al., 2008) and Cu (Shiau and Ning, 2003) were determined for hybrid tilapia O. niloticus x O.

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aureus. The NRC (2011) compiled several studies on trace mineral requirements of fish and the

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supplementation levels suggested for Nile tilapia served as references for the present study. By the time this study was planned there were no information on Se requirement for tilapia. For this

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reason, the recommended dietary level of Se (0.25 ppm) suggested for the channel catfish

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Ictalurus Punctatus by the NRC (2011), as determined by Gatlin and Wilson (1984b), was

for Nile Tilapia at nearly 1 ppm.

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applied in this study. Recently, Lee et al. (2016) have determined the optimum requirement of Se

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Although previous findings indicated that inclusion of trace elements in the diets for several fish and shrimp species had positive effects on the weight gain of targeted animals

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(Apines et al., 2003; Bharadwaj et al., 2014; Katya et al., 2016), no significant effects of trace

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mineral premix level or source were observed on fish growth in this study. One of the possible explanation for this, is the fact that the basal diet used had enough trace elements to sustain

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adequate growth of tilapia. Albeit the basal diet was formulated to have minimum level of indigenous trace minerals derived from the ingredients, the analyzed values (Table 3) of Cu (17.8 - 38.5 mg/kg), Fe (97.1 - 507 mg/kg), Mn (49.8 - 82.4 mg/kg), Zn (56.7 - 190mg/kg) and Se (0.32 - 1.42mg/kg) were all above the requirement recommended by NRC (2011). It’s interesting to notice that all of the ingredients used to formulate the diets were from plants, which are generally considered poor sources of minerals and may contains antagonists, such as phytic acid

ACCEPTED MANUSCRIPT (phytate), which can reduce the bioavailabilty of minerals. In this study, the phytate level in the basal diet was at 0.9%, representing observed concentrations found on plant-based commercial diets. Another possible reason of the successful use of the control diet without micromineral supplements in this study might be because of the acidic condition of the gastric stomach of Nile

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tilapia. This acid condition allows dissociation of the compounds that can be easily absorbed in

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the intestine, contributing to improve the bioavailability of microminerals naturally present in the

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feed ingredients. Also, at lower stomach pH, the phytate molecules have less active adsorption sites, which make minerals more available for absorption (Lynch 1997; Suiryanrayna and

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Ramana, 2015). Similar to the results of this study, several studies conducted on Nile tilapia

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observed no added benefit of supplementing microminerals in fish feed. Sa et al. (2004) didn’t find significant weight improvement of Nile tilapia fed diets containing incremental levels of Zn

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from 25 mg/kg to 400 mg/kg using ZnSO4 H2O as a source of Zn. Eid et al (1994) even found

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that the supplementation of Zn above the requirement (30 mg/kg) had resulted in reduced growth of Nile tilapia. Nevertheless, the supplementation of Fe at 400 mg/kg helped Nile tilapia to get

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better growth performance compared to fish fed Fe free diet and diet with Fe supplemented at

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200 mg/kg (El-Serafy et al., 2007). Hu et al., (2007) also found that the supplementation of Cu at

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30 mg/kg using Cu2+- exchanged montmorillonite significantly improved growth performance of Nile tilapia compared to fish fed the control diet without Cu supplements. In a recent study, Lee et al., (2016) also observed a positive correlation between growth of Nile tilapia and inclusion level of Se. The dietary range of Se (0.3 to 2.06 mg/kg) tested by Lee et al (2016) were comparable to the range observed in the present study (0.32 to 1.32 ppm). As observed in this study, Apines-Amar et al. (2004) was also unable to find significant difference in the growth performance of rainbow trout fed different levels of trace mineral

ACCEPTED MANUSCRIPT premixes supplemented from both sources. Nevertheless, Apines et al. (2003) demonstrated the advantages of using amino acid-chelate as a source of trace element premixes for rainbow trout. In their study, rainbow trout fed semi-purified diets containing tricalcium phosphate and phytate had higher growth rate (TGC) and feed consumption when the diets were supplemented with

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chelated compared to inorganic trace minerals. Apines et al. (2003), however, also mentioned

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that the effect of the mineral chelates on growth was not observed in similar studies using

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practical diets (Apines et al., unpublished). Katya et al., (2016) also demonstrated the added benefit of supplementing chelated trace mineral premixes to meet the requirement of marine

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rockfish Sebastes schlegeli fed on semi-purified diets added of 10 g/kg phytic acid and 20 g/kg

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dicalcium phosphate to mimic the conditions of commercial feeds. Albeit, inorganic trace mineral supplements didn’t help the fish to get better growth performance compared to fish fed

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the basal diet. The supplement of chelated trace mineral to the requirement level of marine

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rockfish helped the fish to attain higher growth rate. In this study, excessive levels of microminerals in the diets did not influence growth,

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neither the survival of fish even though impaired growth rate and high mortality were observed

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in several studies with Nile tilapia. Supplementation of Zn above the requirement (30 mg/kg) have resulted in reduced growth of Nile tilapia (Eid and Ghonim, 1994). Nevertheless, Sa et al

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(2004) fed 13 g Nile tilapia for 70 days with all-plant diets supplemented with graded levels of Zn up to 400 mg/kg and observed no adverse effects on growth and FCR on fish fed the highest Zn diet. Clearwater et al. (2002) also reported the toxic levels of Zn at 9 - 12 mg/kg dietary intakes on Nile tilapia. This toxic intake of Zn would require feeding diets containing nearly 180 to 240 mg Zn/kg at a 5% body weight per day. As this was the feeding rate applied at most part of our study, it can be assumed that the diets with the highest inclusion of minerals (organic

ACCEPTED MANUSCRIPT 4xNRC with 181 ppm of Zn and inorganic 4xNRC with 190 ppm of Zn) had potentially toxic levels of Zn. However, no detrimental effect on tilapia growth, FCR and survival were obtained on Nile tilapia fed the two highest Zn diets in this study. Therefore, it is unlikely that overformulation of Zn at 200 ppm in practical diets would cause toxicity to tilapia, although it

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will result in economic loss and unnecessary waste added to the environment. Regarding Cu, no

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adverse effect of growth and survival were observed on fish fed diets with the highest levels of

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this mineral (38.5 mg Cu/kg, a concentration 10 times higher than recommended by the NRC, 2011). Previous findings of Damasceno et al. (2013) indicated that exposure of Nile tilapia to

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very high dietary levels of Cu of 1,000 and 1,500 ppm had led to a marked depression on growth

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(weight gain of 60 and 40 g) compared to fish fed diets with supplemental 4 to 8 ppm of Cu (122 to 138 g of weight gain in the same period). The survival of fish in their study was also effected

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by excessive dietary Cu in which fish fed diets with 1,500 ppm Cu supplements exhibited

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significantly lower survival (62%) compared to fish fed diets with 4 and 8 ppm of Cu (72% to 87%). The toxicity of Cu has been demonstrated to hybrid tilapia (O. niloticus x O. aureus) at

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lower levels of 20 mg/kg (Shiau and Ning, 2003) in which fish fed diets with 20mg/kg Cu had

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significantly lower growth rate and feed efficiency compared to fish fed diets with other diets supplemented Cu below that. The chronic toxicity of Se was also reported by (Lee et al., 2016) in

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which levels of 14.7 was reported to cause reduced weight gain and survival of fish. Excessive supplements of Mn and Fe has, however, not been seen to cause adverse effects on growth and survival tilapia. El-Serafy et al., (2007) demonstrated that growth and FCR were best at 1,200 ppm of Fe, and observed no deleterious effect on fish performance and survival of Nile tilapia fed diets with up to 1,600 mg/kg Fe. Such value was about 3 times higher than the maximum dietary level of iron observed in the present study (507 mg/kg). No adverse effects on growth,

ACCEPTED MANUSCRIPT feed efficiency and survival were observed on hybrid tilapia fed diet with the highest level of Mn (64.7 ppm) compared to fish fed diets near the optimal level of 7 ppm determined for this mineral. Overall, the high survival (96.8% to 100%) of fish fed practical diets supplemented 4xNRC or diets non-supplemented reinforces that micromineral toxicity did not occurred in this

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study. However, the limited 8-week experimental period of this study has to be taken in

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consideration, as it might not have been long enough to induce sub-lethal or lethal signs of

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mineral toxicity or deficiency.

No significant effect of source or the level of trace minerals were observed on moisture,

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crude protein, lipids and ash contents of tilapia fed different experimental diets in this study. The

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same patterns were observed on ANPR and PER which ranged from 26.0 to 26.9 and 1.64 to 1.72, respectively. Similarly, Eid et al., (1994) and Sa et al., (2004) indicated that no significant

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difference of PER was observed on Nile tilapia fed incremental levels of dietary Zn. Also, El-

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Serafy (2007) demonstrated that the supplementation with increasing levels of Fe had no added benefit on ANPR in Nile tilapia. Nevertheless, the later study conducted by El-Serafy (2013)

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found that fish fed with Cu contaminated diet (2 g/Kg diet) had 39.19% higher protein content

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compared to fish fed the control diet after 30 days of feeding trial. Such level of Cu was 50 times higher than in the present study, suggesting that the dietary Cu levels used in our study were too

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low to cause a change in protein deposition of fish. According to the results of this study, the whole-body retention of the dietary supplied minerals reduced as the dietary supplementation levels increased. Among the trace elements evaluated, Se had the highest retention (22 to 23%) followed by Zn, Cu and Fe. Mn was the mineral with the lowest recorded retention (0.9 to 2.6%). The same pattern was observed in the study of Apines et al., 2003 conducted on rainbow trout, where only a small percentage (5 – 7 %) of Mn was retained in the tissues. In this present study,

ACCEPTED MANUSCRIPT there were also no major differences in the retention of microminerals between fish fed the organic and inorganic sources (P > 0.05). Contrarily, Apines et al., (2001, 2003); Satoh et al., (2001) found that rainbow trout fed amino acid-chelate had higher retention of the elements (Zn, Cu, Mn) compared to the sulfate-supplemented group.

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Tilapia whole body and fillet concentrations of Cu, Mn, Fe, Zn and Se in this study were

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significantly affected by dietary micromineral levels, which agrees with the findings of Eid et al.,

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(1994); Lee at al., (2017) and Sa et al., (2007). However, dietary sources of mineral did not influence the levels of Cu, Mn and Fe contents of Nile tilapia (P>0.05). Similarly, Apines-Amar

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(2014) observed no pronounced effect of inorganic and inorganic sources of minerals on whole

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body Zn and Cu content of rainbow trout. However, fish fed inorganic trace elements had lower Mn concentration compared to fish fed the organic source. Contrary to the results of the present

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study, Katya et al. (2016) suggested a 2 to 4 times higher efficiency of chelated over inorganic

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mineral premix for Cu and Zn saturation. There have also been several reports of lower requirement for chelated over inorganic Cu to promote comparable tissue mineralization (Apines

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et al., 2003; Bharadwaj et al., 2014; Lin et al., 2010).

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Interestingly, pronounced effects of different sources of trace elements were observed on the concentration of Se in the fillet of tilapia. Fish fed diets supplemented with the organic

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source of Se had higher Se concentration in the fillet compared to fish fed diets supplemented with the inorganic source with the adjusted mean values were 0.0197 and 0.0161 mg/kg, respectively (Table 9). At comparable levels of supplementation, the organic trace mineral premix resulted in 11 to 32% higher Se concentrations in the fillet of tilapia, compared to the inorganic one (Fig. 1). US Department Health and Service recommended average daily amounts of Se from 0.015 mg for infant to 0.070 mg for breastfeeding teenagers and women. According

ACCEPTED MANUSCRIPT to the results of our study, the highest Se deposition can be achieved at 0.025 mg/kg fish if the highest level of Se supplement from organic source is to be used (1.14mg Se/kg of diet). Therefore, the sole consumption of 300 g of Se-enriched tilapia fillet per day would be sufficient to meet the highest requirement for daily intake of human. However, U.S Food and Drug

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Administration currently allows a Se supplementation of only 0.3 mg/kg from sodium selenate or

IP

sodium selenite in feeds for all animals, including aquatic species (NRC, 2011). Therefore, more

CR

research is needed to take into consideration of enriched-micromineral products on human health

US

and its cost to the environment.

AN

4. Conclusion

In this study, the supplementation of practical diets with organic or inorganic trace

M

minerals (Cu, Fe, Mn, Se, and Zn) did not further improve growth, feed efficiency and survival

ED

compared to the non-supplemented diet. Practically, all-plant diets seemed to contain enough quantities of these trace elements to satisfy tilapia requirement suggested by the NRC. This

PT

finding suggested that the overdosing of trace elements in practical diets should be reconsidered

CE

in order to avoid unnecessary feed costs and excessive discharge of trace elements to the environment. However, it should be stated that the controlled experimental lab conditions

AC

applied in this experiment are considerably different than the challenges imposed by environmental conditions and management in intensive commercial aquaculture systems used for tilapia production. In addition, the time required for producing a marketable size tilapia ranges from 4 to 8 months, which is much longer than the 8-week period of this study. Therefore, it is possible that trace mineral supplementation to practical diets brings additional benefits to production performance and health of tilapia in intensive managed systems. Finally, the Se

ACCEPTED MANUSCRIPT contents of tilapia fillet can be effectively enriched by adjusting dietary doses of this trace element. This can make farmed tilapia products an effective and desirable source of Se for human nutrition.

T

Acknowledgment

IP

The authors would like to express our gratitude and appreciation to those who have taken

CR

the time to critically review this manuscript as well as those who helped support this research at the E.W. Shell Research Station, School of Fisheries, Aquaculture and Aquatic Sciences, Auburn

US

University. Special thanks to students who helped maintain the daily management during the

AN

trials. The work was supported in part by grants from Altech and the Alabama Agricultural Experiment Station and the Hatch program (ALA016-08027) of the National Institute of Food

M

and Agriculture, U.S. Department of Agriculture. The mention of trademarks or proprietary

ED

products does not constitute an endorsement of the product by Auburn University and does not

AC

CE

PT

imply its approval to the exclusion of other products that may also be suitable.

ACCEPTED MANUSCRIPT References Apines-Amar, M.J.S., Satoh, S., Caipang, C.M.A., Kiron, V., Watanabe, T., Aoki, T., 2004. Amino acid chelates: a better source of Zn, Mn and Cu for rainbow trout Oncorhynchus mykiss. Aquaculture 240, 345–358.

T

Apines, M.J., Satoh, S., Kiron, V., Watanabe, T., Nasu, N., Fujita, S., 2001. Bioavailability of

IP

amino acids chelated and glass embedded zinc to rainbow trout, Oncorhynchus mykiss,

CR

fingerlings. Aquac. Nutr. 221–228.

Apines, M.J.S., Satoh, S., Kiron, V., Watanabe, T., Aoki, T., 2003. Availability of supplemental

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amino acid-chelated trace elements in diets containing tricalcium phosphate and phytate

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to rainbow trout, Onocrhynchus mykiss. Aquaculture 225, 431–444. Ashmead, H.D., 1992. Factors which affect the intestinal absorption of minerals. In: The Role of

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Amino Acid Chelates in Animal Nutrition. Noyes Publications, Park Ridge, NJ, USA,

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Barros, M.M., Pezzato, L.E., Kleemann, G.K., Hisano, H., Rosa, G.J.M., 2002. Levels of

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ACCEPTED MANUSCRIPT Chatvijitkul, S., 2015. Chemical composition and pollution potential of fish and shrimp feeds. PhD dissertation. Auburn University, AL, USA, pp. 115-136. Clearwater, S.J., Farag, A.M., Meyer, J.S., 2002. Bioavailability and toxicity of dietborne copper and zinc to fish. Comp. Biochem. Physiol. 132, 269-313.

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Damasceno, F.M., Teixeira, C.P., Silva, R.L., Rocha, M.K.H.R., Fernandes Júnior, A.C., Barros,

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Davis, D.A., 2015. Feed and feeding practices in Aquaculture. Woodhead Publishing Series in

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Davis, D.A., Lawrence, A.L., Gatlin III, D.M., 1993. Dietary copper requirement of Penaeus vannamei. Nippon Suisan Gakkaishi 59, 117–122.

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Eid, A.E., Ghonim, S.I., 1994. Dietary zinc requirement of fingerling Oreochromis niloticus.

El-Serafy, S.S., El-Ezabi, M.M., Shehata, T.M., Esmael, N., 2007. Dietary iron requirement of

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the Nile tilapia, Oreochromis niloticus (L.) fingerlings in intensive fish culture. Egypt. J.

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FAO Fisheries and Aquaculture Department, Food and agriculture organization of the United Nations, Rome. Fernandes, D., Zanuy, S., Bebianno, M.J., Porte, C., 2008. Chemical and biochemical tools to assess Pollut. exposure in cultured fish. Env. Pollut. 152, 138-146. Fitzsimmon, K., 2016. Supply and demand in global tilapia markets 2015. WAS 2016, Las Vegas, USA.

ACCEPTED MANUSCRIPT Garcia-Aranda, J.A., Wapnir, R.A., Lifshitz, F., 1983. In vivo intestinal absorption of manganese in the rat. J. Nutr. 113, 2601–2607. Gatlin III, D. M., and Wilson, R. P., 1984. Dietary selenium requirement of fingerling channel catfish. J. nutr. 114, 627-633.

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Gatlin III, D. M., and Willson, R. P., 1986. Characterization of iron deficiency and the dietary

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iron requirement of fingerling channel catfish. Aquaculture 52, 191-198.

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Hardy, R.W., Shearer, K.D., 1985. Effects of dietary calcium phosphate and zinc supple-

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mentation on whole body zinc concentrations of rainbow trout (Salmo gairdneri). Can. J. Fish. Aquat. Sci. 42, 181–184.


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Hill, A.D., Peo E.R.Jr, Lewis, A.J., Crenshaw, J.D., 1986. Zinc-amino acid complexes for swine. J. Anim. Sci. 63, 123–130.

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Hu, C.H., Xu, Y., Xia, M.S., Xiong, L., Xu, Z.R., 2007. Effects of Cu2+-exchanged

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montmorillonite on growth performance, microbial ecology and intestinal morphology

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of Nile tilapia (Oreochromis niloticus). Aquaculture 270, 200-206. Katya, K., Lee, S., Yun, H., Dagoberto, S., Browdy C.L., Vazquez-Anon, M., Bai, S.C., 2016.

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Efficacy of inorganic and chelated trace minerals (Cu, Zn and Mn) premix sources in

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Pacific white shrimp, Litopenaeus vannamei (Boone) fed plant protein based diets. Aquaculture 459, 117–123. Katya, K., Lee, S., Bharadwaj, A.S., Browdy, C.L,
Vazquez-Anon, M., and Bai, S.C., 2016. Effects of inorganic and chelated trace mineral (Cu, Zn, Mn and Fe) premixes in marine rockfish, Sebastes schlegeli (Hilgendorf), fed diets containing phytic acid. Aquac. Res. 48, 4165-4173.

ACCEPTED MANUSCRIPT Lall, S.P., 2002. The minerals. In: Fish Nutrition. J.E. Halver and R.W. Hardy (eds.), 3rd edition. London: Academic Press. pp. 259-308. Lee. S., Nambi, R.W., Won, S., Katya, K., Bai, S.C., 2016. Dietary selenium requirement and toxicity levels in juvenile Nile tilapia, Oreochromis niloticus. Aquaculture 464, 153-158.

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Lin, Y.H., Lin, S.M., and Shiau, S.Y., 2008. Dietary zinc requirements of juvenile tilapia,

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Oreochromis niloticus x O. aureus. J. Fish. Soc. Taiwan. 35, 117-125.

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Lin, Y.H., Lin, S.M., Shiau, S.Y., 2008b. Dietary manganese requirements of juvenile tilapia, Oreochromis niloticus x Oreochromis aureas. Aquaculture 284, 207-210.

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Moriarty, D.J.W., 1973. The physiology of digestion of blue-green algae in the cichlid

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fish Tilapia nilotica. J. Zool. Lond. 171, 25-39.

Paripatananont, T., Lovell, R.T., 1995a. Chelated zinc reduces the dietary zinc requirement of

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channel catfish, Ictalurus punctatus. Aquaculture 133, 73–82.

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Paripatananont, T., Lovell, R.T., 1995b. Responses of channel catfish fed organic and inorganic

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sources of zinc to Edwardsiella ictaluri challenge. J. Aquat. Anim. Health 7, 147–154.
 Paripatananont, T., Lovell, R.T., 1997. Comparative net absorption of chelated and inorganic

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62–67.


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trace minerals in channel catfish Ictalurus punctatus diets. J. World Aquacult. Soc. 28,

Puchala, R., Sahlu, T., Davis, J.J., 1999. Effects of zinc-methionine on performance of Angora goats. Small Rumin. Res. 33(1), 1–8. Satoh, S., Apines, M.J., Tsukioka, T., Kiron, V., Watanabe, T., Fujita, S., 2001. Bioavailability of amino acids chelated and glass embedded manganese to rainbow trout, Onchorhynchus mykiss, fingerlings. Aquac. Res. 32S, 18–25. Scott, M.L., Nesheim, M.C., Young, R.J., 1982. Nutrition of the Chicken Scott, M.L. and

ACCEPTED MANUSCRIPT Associates, Ithaca, NY. Shiau, S.Y., Ning, Y.C., 2003. Estimation of dietary copper requirements of juvenile tilapia, Oreochromis niloticus × O. aureus. Anim. Sci. 77, 287–292. Tan, B., Mai, K., 2001. Zinc methionine and zinc sulfate as sources of dietary zinc for juvenile

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abalone, Haliotis discus hannai Ino. Aquaculture 192, 67–84.

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Watanabe, T., Satoh, S., and Takeuchi, L., 1988. Trace mineral in fish nutrition. Aquaculture

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151, 185-207.

Wedekind, K.J., Baker, D.H., 1989. Zinc bioavailability in feed-grade zinc sources. J. Anim. Sci.

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67S, 126.

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Wedekind, K.J., Hortin, A.E., Baker, D.H., 1992. Methodology for assessing zinc bioavailability:

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CE

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ED

M

efficacy for ZnMet, zinc sulfate and zinc oxide. J. Anim. Sci. 70(1), 178–187.

ACCEPTED MANUSCRIPT Table 1. Source and composition of the inorganic and organic micromineral premixes used in the experimental diets. Grams % mineral in the Source

Mineral

Mineral form

source/100g source

Zinc sulphate.7H2O

22.7

CR

Manganous sulphate.H2O

Cu

Cupric sulphate.5H2O

25.5

1.57

Fe

Ferrous sulphate

20.1

42.31

Se

Sodium selenite

43.0

0.06

I

Potassium Iodidea

76.4

0.13

M

AN

US

32.5

13.19

Mn

Inorganic

2.15

40.59

Zn – Organicb

15.0

20.00

Mn

Mn – Organicb

15.0

4.67

Cu – Organicb

12.0

3.33

Fe – Organicb

15.0

56.67

Se – Organicc

0.3

8.33

Potassium Iodidea

76.4

0.13

PT

Zn

ED

Cellulose

Fe

AC

Se

CE

Cu Organic

IP

Zn

T

premix

I

Cellulose

6.87

a

Potassium iodide was used as the Iodide source in both premixes.

b

Originated from partial hydrolysis of soybean proteins reacted with metal salt (Alltech®)

c

Selenium enriched yeast (Selplex, Alltech®)

of

ACCEPTED MANUSCRIPT Table 2. Ingredient compositions (g 100 g-1 as-is) of nine experimental diets with increasing levels of micromineral supplement from inorganic or organic sources offered to juvenile Nile tilapia (7.13 ± 0.24) in 8-weeks.

O-1.0

O-2.0

O-4.0

I-0.5

I-1.0

Soybean meala

53.80

53.80

53.80

53.80

53.80

53.80

Whole wheatb

15.00

15.00

15.00

15.00

15.00

concentratec

9.70

9.70

9.70

9.70

Soybean oild

4.30

4.30

4.30

Wheat floure

5.00

5.00

5.00

Yellow cornf

8.35

8.35

Vitamin premixg

0.50

0.50

Choline chlorideh

0.05

0.05

Stay C 35%i

0.10

Salt (NaCl)j

0.50

53.80

53.80

15.00

15.00

15.00

15.00

9.70

9.70

9.70

9.70

9.70

4.30

4.30

4.30

4.30

4.30

4.30

5.00

5.00

5.00

5.00

5.00

5.00

8.35

8.35

8.35

8.35

8.35

8.35

8.35

0.50

0.50

0.50

0.50

0.50

0.50

0.50

0.05

0.05

0.05

0.05

0.05

0.05

0.05

0.10

0.10

0.10

0.10

0.10

0.10

0.10

0.10

0.50

0.50

0.50

0.50

0.50

0.50

0.50

0.50

2.00

2.00

2.00

2.00

2.00

2.00

2.00

2.00

2.00

0.30

0.30

0.30

0.30

0.30

0.30

0.30

0.30

0.30

0.05

0.10

0.20

0.40

0.05

0.10

0.20

0.40

0.35

0.30

0.20

PT

AC

Methioninel

CE

CaP-dibasick

M

IP

CR

premix

inorganicn Cellufillh

US

premix

organicm Mineral

53.80

protein

I-2.0

T

O-0.5

Mineral

ol

AN

Ingredients

Corn

I-4.0

ED

Contr

0.35

0.30

0.20

0.40

ACCEPTED MANUSCRIPT Supplemented levels of micro minerals (as ppm) 42.50

85.00

170.00 340.00 42.50

85.00

170.00 340.00

Manganese

3.50

7.00

14.00

28.00

3.50

7.00

14.00

28.00

Copper

2.00

4.00

8.00

16.00

2.00

4.00

8.00

16.00

Zinc

15.00

30.00

60.00

120.00 15.00

30.00

60.00

120.00

Selenium

0.13

0.25

0.50

1.00

0.25

0.50

IP

0.13

T

Iron

De-hulled solvent extract soybean meal, Bunge Limited, Decatur, AL, USA

b

Bob’s Red Mill Natural Foods, Milwaukie, OR, USA

c

Empyreal® 75, LystoTM, Cargill Corn Milling, Cargill, Inc., Blair, NE, USA

d

Vigon®, East Stroudsburg, PA, USA

e

King Arthur Flour, Norwich, Vermont, USA

f

Faithway Feed Co., Guntersville, AL, USA.

M

AN

US

CR

a

1.00

g

ED

Vitamin (g/kg premix): Thiamin HCl, 0.44; Riboflavin, 0.63; Pyridoxine HCl, 0.91; DL

pantothenic acid, 1.72; Nicotinic acid, 4.58; Biotin, 0.21; Folic acid, 0.55; Inositol, 21.05;

PT

Menadione sodium bisulfite, 0.89; Vitamin A acetate, 0.68; Vitamin D3, 0.12; dL-alpha-

CE

tocoperol acetate, 12.63; Alpha-cellulose, 955.59 MP Biochemicals Inc., Solon, OH, USA

i

Stay C®, (L-ascorbyl-2-polyphosphate 25% Active C), DSM Nutritional Products., Parsippany,

NJ, USA.

AC

h

j

Carolina Biological Supply Company, Burlington, NC, USA

k

Alfa Aesar, Ward Hill, MA, USA

l

TCI, Tokyo, Japan

m

Alltech®, Springfield, KY, USA

ACCEPTED MANUSCRIPT

CE

PT

ED

M

AN

US

CR

IP

T

Inorganic mineral premixs (Formula is presented in Table 1)

AC

n

ACCEPTED MANUSCRIPT Table 3. Proximate composition (% in original matter) and mineral composition1 (g kg-1: calcium, magnesium, phosphorus, potassium, sodium; sulfur, mg kg-1: copper, iron, manganese,

O-0.5

O-1.0

O-2.0

O-4.0

I-0.5

I-1.0

I-2.0

I-4.0

Control

Moisture

7.24

4.72

7.18

8.08

8.38

8.64

8.49

7.74

8.13

Dry matter

92.76

95.28

92.72

91.92

91.62

91.36

91.51

92.26

91.87

Crude protein

36.90

37.80

37.20

36.60

36.80

36.40

36.20

37.00

36.90

Fat

5.44

5.48

4.67

4.78

4.95

4.86

5.38

5.30

5.68

Fiber

7.90

6.20

7.60

7.40

Ash

5.80

5.90

5.90

5.90

Calcium

0.95

0.99

0.95

0.94

Magnesium

0.18

0.18

0.18

Phosphorus

0.85

0.88

0.86

Potassium

1.26

1.28

Sodium

0.25

0.26

Sulfur

0.41

Copper

17.80

Iron

139.00 177.00 280.00 404.00 128.00 184.00 277.00 507.00 97.10

AC

CR

6.80

7.60

7.40

5.90

5.80

5.70

5.90

5.70

0.90

AN

0.97

0.92

0.98

1.00

0.18

0.17

0.18

0.18

0.19

0.19

0.85

0.84

0.87

0.85

0.91

0.92

ED

21.10

US

8.30

1.28

1.24

1.20

1.28

1.27

1.35

1.38

0.25

0.25

0.24

0.25

0.25

0.27

0.27

0.42

0.42

0.40

0.41

0.42

0.46

0.44

31.00

35.00

22.60

23.30

33.50

38.50

19.60

M

9.00

PT

CE

0.42

IP

Treatment

T

selenium, zinc) of the test diets

Manganese

49.80

55.60

64.40

77.30

48.20

54.30

61.70

82.40

49.90

Selenium

0.39

0.53

0.65

1.14

0.46

0.59

0.77

1.42

0.32

Zinc

67.70

87.40

125.00 181.00 66.10

82.90

115.00 190.00 56.70

1

Diets were analyzed at Midwest Laboratories (Omaha, NE, USA).

ACCEPTED MANUSCRIPT Table 4. Mean values1 for the initial and final weight (IW and FW), percent weight gain (WG), thermal growth coefficient (TGC), feed conversion ratio (FCR), survival, apparent net protein retention (ANPR) and protein efficiency ratio (PER) of Nile Tilapia (7.13 ± 0.24) fed diets supplemented with increased levels of organic or inorganic microminerals, after an 8-week

T

feeding period.

IW(g)

FW (g)

WG (%) TGC

FCR

IP

Survival

Treatment

ANPR

PER

98.4%

26.5

1.71

7.1

38.8

446.78

0.0968

O-1.0

7.0

38.9

454.54

0.0975

1.56

98.4%

26.6

1.70

O-2.0

7.1

38.4

437.86

0.0957

1.64

96.8%

26.0

1.64

O-4.0

7.1

39.1

450.16

0.0973

99.2%

26.4

1.70

I-0.5

7.1

38.1

439.93

AN

1.60

0.0956

1.64

96.8%

26.5

1.66

I-1.0

7.2

38.7

435.66

0.0959

1.65

97.6%

26.6

1.67

I-2.0

7.2

39.1

440.98

0.0965

1.61

99.2%

26.9

1.72

I-4.0

7.2

38.6

436.04

0.0958

1.65

98.0%

26.1

1.64

Control

7.1

433.63

0.0950

1.60

100.0%

26.8

1.70

P-value

0.846

0.461

0.802

0.662

0.451

0.770

0.987

0.137

PSE2

0.116

0.441

9.539

0.001

0.021

1.412

0.607

0.023

Source

0.329

0.547

0.209

0.245

0.527

0.778

0.729

0.451

Level

0.806

0.795

0.949

0.940

0.910

0.924

0.965

0.944

Source*Level 0.611

0.427

0.708

0.607

0.180

0.538

0.811

0.233

M

PT

CE

AC

Two-way

37.9

1.59

US

O-0.5

ED

CR

(%)

ANOVA

ACCEPTED MANUSCRIPT 1

Means (n=5 for all treatments except treatment I-4.0 with n = 4) in the same column are not

significantly different at P > 0.05 based upon analysis of variance followed by Tukey’ multiple range test.

CE

PT

ED

M

AN

US

CR

IP

T

Pooled standard error.

AC

2

ACCEPTED MANUSCRIPT Table 5. Mean values1 (as % of original matter) of whole body proximate composition of Nile Tilapia (7.13 ± 0.24) fed diets supplemented with increased levels of organic or inorganic microminerals, after an 8-week feeding period. Crude protein

Moisture

Lipids

Ash

O-0.5

15.27

73.81

7.12

3.48

O-1.0

15.35

73.77

6.69

O-2.0

15.60

73.39

6.85

O-4.0

15.25

73.56

I-0.5

15.60

73.86

I-1.0

15.60

74.04

I-2.0

15.41

I-4.0

CR

IP

T

Treatment

US

7.24

3.59 3.49 3.59 3.74

6.57

3.52

73.37

7.18

3.57

15.60

72.47

7.92

3.60

Control

15.46

73.87

6.58

3.67

P-value

0.957

0.662

0.677

0.885

PSE2

0.257

0.509

0.48

0.127

0.339

0.698

0.829

0.402

0.989

0.353

0.349

0.899

0.748

0.595

0.727

0.609

AC

ANOVA

Level

Source*Level 1

M

ED

PT

CE

Two-way

Source

AN

6.73

Means (n=5 for all treatments except treatment I-4.0 with n = 4) in the same column are not

significantly different at P > 0.05 based upon analysis of variance followed by Tukey’ multiple range test.

ACCEPTED MANUSCRIPT

CE

PT

ED

M

AN

US

CR

IP

T

Pooled standard error.

AC

2

ACCEPTED MANUSCRIPT Table 6. Mean values1 of whole body micromineral composition (in ppm or mg kg-) of Nile Tilapia (7.13 ± 0.24) fed diets supplemented with increased levels of organic or inorganic microminerals, after an 8-week feeding period. Cu

Fe

Mn

Se

Zn

O-0.5

2.68b

18.09b

1.66b

0.21de

24.40ab

O-1.0

3.00ab

20.12ab

1.58b

0.27cd

24.81ab

O-2.0

3.03ab

19.59ab

1.28b

0.27cd

26.15ab

O-4.0

3.34ab

21.61a

1.55b

0.38b

25.71ab

I-0.5

2.75ab

20.24ab

2.03a

0.22de

26.80a

I-1.0

3.11ab

19.20ab

1.44b

0.24de

23.58b

I-2.0

3.01ab

19.87ab

1.44b

0.33bc

24.74ab

I-4.0

3.77a

21.64a

1.34b

0.53a

26.04ab

Basal

2.62b

17.42b

2.00a

0.18e

24.44ab

P-value

0.031

0.006

<0.0001

<0.0001

0.036

PSE2

0.215

0.748

0.091

0.02

0.666

0.457

0.573

0.332

0.021

0.974

0.007

0.022

<0.0001

<.0.0001

0.101

0.757

0.265

0.008

0.002

0.034

Level

AC

ANOVA Source

Source*Level 1

IP CR

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ED PT

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Two-way

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Treatment

Means (n=5 for all treatments except treatment I-4.0 with n = 4) in the same column with

different superscripts are significantly different at P < 0.05 based upon analysis of variance followed by Tukey’ multiple range test.

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ACCEPTED MANUSCRIPT Table 7. Mean values1 of whole body mineral retention (%) of Nile tilapia fed diets supplemented with increased levels of organic or inorganic microminerals, after an 8-week

Cu

Fe

Mn

Se

Zn

O-0.5

10.04a

7.92c

2.04b

32.80

23.08c

O-1.0

9.88a

7.26c

1.75bc

34.05

18.60d

O-2.0

6.46b

4.20e

1.08de

26.34

13.14e

O-4.0

6.49b

3.36ef

1.19de

22.51

9.07f

I-0.5

7.94ab

9.60b

2.64a

29.64

25.71b

I-1.0

8.85ab

6.21d

1.51cd

24.83

17.46d

I-2.0

6.01b

4.41e

1.35cde

28.09

13.61e

I-4.0

6.59b

2.60f

0.89e

25.66

8.55f

Control

8.85ab

10.72a

2.56a

31.73

27.45a

P-value

0.0001

<0.0001

<0.0001

0.033

<0.0001

PSE

0.676

0.349

0.131

2.502

0.508

0.081

0.458

0.176

0.352

0.039

<0.0001

<0.0001

<0.0001

0.061

<0.0001

0.390

0.002

0.002

0.114

0.004

Level

AC

ANOVA Source

Source*Level 1

IP CR

US

M

ED PT

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Two-way

T

Treatment

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feeding period.

Means (n=5 for all treatments except treatment I-4.0 with n = 4) in the same column with

different superscripts are significantly different at P < 0.05 based upon analysis of variance followed by Tukey’ multiple range test.

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ACCEPTED MANUSCRIPT Table 8. Mean concentration1 of microminerals in the fillet (mg 100 g-1 as-is) of Nile Tilapia (7.13 ± 0.24) fed diets supplemented with increased levels of organic or inorganic

Cu

Fe

Mn

Se

Zn

O-0.5

0.057

0.530

0.007

0.015c

0.676

O-1.0

0.052

0.530

0.012

0.018bc

0.666

O-2.0

0.046

0.506

0.007

0.020b

0.632

O-4.0

0.045

0.530

0.005

0.025a

0.628

I-0.5

0.068

0.520

0.012

0.015c

0.640

I-1.0

0.055

0.542

0.016

0.015c

0.652

I-2.0

0.048

0.544

0.011

0.018bc

0.638

I-4.0

0.062

0.538

0.000

0.019b

0.645

Control

0.051

0.460

0.010

0.015c

0.646

P-value

0.350

0.745

0.864

<0.0001

0.934

PSE2

0.007

0.033

0.007

0.001

0.026

0.138

0.607

0.609

0.001

0.721

0.229

0.977

0.440

<0.0001

0.676

0.717

0.899

0.872

0.085

0.770

Level

AC

ANOVA Source

Source*Level 1

IP CR

US

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ED PT

CE

Two-way

T

Treatment

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microminerals, after an 8-week feeding period.

Means (n=5 for all treatments except treatment I-4.0 with n = 4) in the same column with

different superscripts are significantly different at P < 0.05 based upon analysis of variance followed by Tukey’ multiple range test.

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ACCEPTED MANUSCRIPT Table 9. Analysis of covariance output of micominerals concentration in the fillet (mg 100 g-1 as-is) of Nile tilapia (7.13 ± 0.24) fed diets with increasing levels of mineral supplement from organic and inorganic sources over a ten-week growth period.

Mineral source

0.524 0.536 0.999

0.650 0.644 0.624

0.0197a 0.0161b 0.273

Intercept P value

0.893

0.539

<0.0001

CR

1

IP

Organic Inorganic Slope P value

T

Adjusted mean least-squares means of fillet mineral contents Iron Zinc Selenium Copper

1

0.049 0.059 0.921 0.170

Means (n=5 for all treatments except treatment I-4.0 with n = 4) in the same column with

PT

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different superscripts are significantly different at P < 0.05 based upon analysis of covariance.

0.030

CE

0.020

y = 0.0043x + 0.0135 R² = 0.5918

AC

Se deposition (mg 100 g-1 )

0.025

y = 0.012x + 0.0112 R² = 0.7571

0.015

Organic Inorganic

0.010 0.005 -

0.20

0.40

0.60

0.80

1.00

Dietary Se (%)

1.20

1.40

1.60

ACCEPTED MANUSCRIPT Fig. 1. Regression of mean Se deposition of Nile tilapia against dietary Se levels. Solid line represents Se concentration in the fillet of Nile tilapia fed diets containing organic source of Se; dashed line is Se concentration in the fillet of Nile tilapia fed diets containing inorganic source of

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Se.

ACCEPTED MANUSCRIPT Highlights 

The study was conducted using Nile tilapia to evaluate the effects of different levels of traditional inorganic and commercial chelated (Alltech®) sources of trace minerals (Cu, Zn, Fe, Mn and Se) on the growth performance of Nile Tilapia. Results indicated that no significant effects of trace mineral premix levels and sources

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were observed on the growth performance, survival rate and whole body proximate

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Pronounced effects of different sources of trace elements were observed on the concentration of Se in the fillet of tilapia. Fish fed diets supplemented with the organic

AN

source of Se had higher Se concentration in the fillet compared to fish fed diets

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supplemented with the inorganic source.

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composition of Nile tilapia (P > 0.05).