Effects of dietary Macsumsuk^® supplementation on growth performance, haematological parameters, disease resistance and body composition of juvenile Nile tilapia, Oreochromis niloticus L.

Abstract

An eight-week feeding trial was conducted to evaluate the effects of dietary Macsumsuk^® supplementation on growth performance, haematological parameters, disease resistance and body composition of juvenile Nile tilapia, Oreochromis niloticus. A total of 375 fish averaging 1.0 ± 0.05 g (mean ± SD) were randomly distributed into 15 tanks in groups of 25, and each tank was then randomly assigned to one of three replicates of five diets containing 0(M₀), 0.3(M_0.3), 0.6(M_0.6), 1.2(M_1.2) and 2.4(M_2.4)% dietary Macsumsuk^®. At the end of eight weeks of feeding trial, average weight gain (WG), specific growth rate (SGR), feed efficiency (FE) and protein efficiency ratio (PER) of fish fed M_1.2 diet were significantly higher than those fed M₀ diet (P < 0.05). Significantly higher values for haematological parameters, such as red blood cell (RBC), haemoglobin (Hb) and haematocrit (HCT), were obtained in fish fed M_0.6 diet compared to the fish offered M_2.4 diet. Although an analysis of variance (ANOVA) test suggested that the optimum level of dietary Macsumsuk^® in juvenile Nile tilapia, O. niloticus, could be 1.2%, broken-line analysis of WG indicated a level of 0.803% when Macsumsuk^® is used as the dietary feed additive.

Keywords:

• growth promoter
• body composition
• feed additive
• Edwardsiella tarda

1. Introduction

In recent years, aquaculture industry has developed rapidly in many countries. The aquaculture production improves by 15% annually and is predicted to continue to grow fast in future (Dadgar et al. 2010). The sustainability of aquaculture development is absolutely dependent on the availability of quality and cheap feedstuffs. Feed costs generally account for more than half of operating costs of aquaculture operations (Cho & Slinger 1979). Any reduction in feed costs would have a direct positive effect on profitability of aquaculture (Francis et al. 2005; Henry & Alexis 2009). The feed additives improve the immunity, productivity and economic efficiency of fish via its improvement body weight (BW) of the fish (Carnevali et al. 2006), weight gain (WG; Venkat et al. 2004), feed conversion ratio and efficiency (Abdel Hamid & Mohamed 2008). For all these reasons and the effect of feed additives into the fish ration, the returns and net profit will be improved with minimization of the fish production costs (Abdel Hamid & Mohamed 2008; Henry & Alexis 2009). Consequently, numerous studies have investigated the efficacy of various additives in aquafeeds that could be used to improve the performance of fish: plant products such as herbs (Yin et al. 2009), root (Sharma et al. 2010), seed meal (Ahmad & Abdel-Tawwab 2011), onion (Cho & Lee 2012) and green tea (Hwang et al. 2013); macroalgae such as Ulva pertusa (Satoh et al. 1987) and Undaria (Park et al. 2003); microalgae such as Chlorella ellipsoidea (Kim et al. 2002) and Dunaliella (Supamattaya et al. 2005); chitosan (Cha et al. 2008); and yeast glucan (Baulny et al. 1996). However, more studies to develop dietary additives to improve the performance of fish in fish farming are required.

Macsumsuk^® is a kind of rare earth mineral that radiates more than 90% of eco-friendly beneficial far infrared rays in a range of 8–10 µm, a bio-absorbable wavelength range. Macsumsuk^® contains abundant natural minerals such as silica (SiO₂), aluminium (Al), calcium (Ca), magnesium (Mg), ferrous (Fe) and various other beneficial material. Because of this, it is very beneficial to bioactivity. Refined Macsumsuk^® through sorting, firing, burning and grinding processes has been routinely used for medical products, medicine and purification of water and air. Macsumsuk^® has also been reported to have a strong antibacterial effect, vitalize the cells, help promote the growth and development and boost the immunity against diseases (Macsumsuk GM, CO., LTD 2012).

Nile tilapia, Oreochromis niloticus (L.) is one of the most economically important species in aquaculture to culture because of its rapid growth, good survival in high density culture and disease tolerance (El-Sayed 2006). However, currently no reports are available regarding the effect of Macsumsuk^® as a feed additive on fish growth. Therefore, this study aimed at elucidating the potential effects of Macsumsuk^® on growth performance, feed utilization, blood parameters and disease resistance of juvenile Nile tilapia challenged with Edwardsiella tarda.

2. Materials and methods

2.1. Preparation of experimental diet

Dietary additive Macsumsuk^® powder (Table 1) was provided by the Macsumsuk^® GM company, Republic of Korea. Five experimental diets were formulated to be isonitrogenous and isoenergetic and to contain 42.0% crude protein and 15.9 kJ available energy/g diet (NRC 2001). Dietary Macsumsuk^® was supplemented at 0(M₀), 0.3(M_0.3), 0.6(M_0.6), 1.2(M_1.2) and 2.4(M_2.4)% in place of cellulose. Fish meal and dehulled soybean meal were used as the protein sources, soybean oil and fish oil as the lipid sources, and wheat flour and corn starch as the carbohydrate sources in the experimental diets. Dietary formulation and proximate composition of the basal diet are shown in Table 2.

Table 1. Mineral composition of Macsumsuk.

	Macsumsuk (%)
Ca	9.56
P	0.24
K	2.35
Na	4.21
Mg	2.50
Fe	10.53
Zn	0.87
Cu	0.01
Mn	0.41

Note: Results reported on the dry matter basis, average of triplicate. Macsumsuk proximate composition – ash: 99.11%; moisture: 0.12%; no protein; no lipid.

Table 2. Dietary formulation and proximate composition of the basal diet for juvenile Nile Tilapia, Oreochromis niloticus.^a

Ingredients (% DM)	Basal diet
Fish meal^b	34.8
Dehulled soybean meal^b	30.0
Wheat flour^b	18.0
Corn starch^b	6.3
Soybean oil^b	2.5
Fishoil^b	4.0
Vitamin premix^c	1.0
Mineral premix^d	1.0
Cellulose^e	2.4
Macsumsuk^f	0.0
Proximate analysis
Moisture (%)	6.81
Crude protein (%)	42.57
Crude lipid (%)	10.30
Crude ash (%)	11.24

^a Results reported on the dry matter basis, average of triplicate.

^b Suhyup Feed Co., Uiryeong, Korea.

^c Contains (as mg kg⁻¹ in diets): ascorbic acid, 300; dl-calcium pantothenate, 150; choline bitate, 3000 L inositol, 150; menadione, 6; niacin, 150; pyridoxine HCl, 15; riboflavin, 30; thiamine mononitrate, 15; dl-α-tocopherol acetate, 201; retinyl acetate, 6; biotin, 1.5; folic acid, 5.4; cobalamin, 0.06.

^d Contains (as mg kg⁻¹ in diets): NaCl, 437.4; MgSO₄·7H₂O, 1379.8; ZnSO₄·7H₂O, 226.4; Fe-citrate, 299; MnSO₄, 0.016; FeSO₄, 0.0378; CuSO₄, 0.00033; calcium iodate, 0.0006; MgO, 0.00135; NaSeO₃, 0.00025.

^e Sigma-Aldrich Korea Yongin, Korea: the amount of each diet would be adjusted by the level of Macsumsuk^®.

^f Macsumsuk^® GM.

Procedures for diet preparation and storage were followed as previously described by Bai and Kim (1997). Diets were prepared by mixing the dry ingredients in an electric mixer, followed by the addition of oil and water. This mixture was formed into dough and dry pellets were made by passing the dough through a screw-type pelleting machine and air drying the formed pellets for approximately 48 h. After drying, the pellets were broken up, sieved into the proper diameter size, sealed and stored at −20°C until use for feeding trial.

2.2. Experimental design

The feeding trial was carried out at the Feeds and Foods Nutrition Research Center, Pukyong National University, Busan, South Korea. Fish were transported to the experimental station and acclimated to the experimental conditions for one week before the feeding trial began. At the start of the experiment, 25 Nile tilapia with an initial weight averaging 1.0 ± 0.05 g (mean ± SD) were randomly distributed into each of the 15 tanks. Each tank was then randomly assigned to one of three replicates of five dietary treatments. Fish were fed twice daily (09:00 and 18:00 h) for eight weeks at a fixed rate of 5% of wet BW/day. Total fish weight in each tank was determined every two weeks and the feeding rate was adjusted accordingly. The feeding trial was conducted by using the semi-recirculating system with 15 tanks (40 L) receiving filtered seawater at the rate of 0.8 L/min from the centre tank. Supplemental aeration was provided to maintain the dissolved oxygen at 6.0 ± 0.2 mg L⁻¹, and also water temperature, ammonia nitrogen, nitrite and pH during the experiment were maintained at 26 ± 0.5°C, 0.05 ± 0.01 mg NH₄-N/L, 0.13 ± 0.05 mg NO₂-N/L and 8.13 ± 0.13, respectively. Siphoning was done every morning in each rearing tank; also, dead fish were removed, weighed and recorded daily.

2.3. Sample collection and analysis

At the end of the feeding trial, fish were weighed and counted to determine WG, feed efficiency (FE), specific growth rate (SGR), protein efficiency ratio (PER) and survival. After the final weighing, blood samples were obtained from the caudal vessels of three individual fish per tank (nine fish per treatment group) by using a heparinized syringe and pooled into treatment groups for blood analysis. Haematocrit (HCT; packed cell volume, PCV) was determined by the micro HCT e method (Brown 1980), and haemoglobin (Hb) was measured with the same fish by the cyanmethaemoglobin procedure using Drabkin's solution. Hb standard prepared from human blood (Sigma Chemical Co., St Louis, MO, USA) was used.

Five fish were freeze-dried as whole body and held at −20°C until used for proximate composition analysis. The tested diets and whole-fish body from each treatment were analysed according to the standard methods of AOAC (2000) for moisture, protein, lipid and ash. Samples of diet and fish were dried at 135°C for 2 h to a constant weight to determine their moisture content. Ash content was determined by incineration at 600°C. Crude protein was determined using the Kjeldahl method (N × 6.25) after acid digestion, and crude lipid was determined by soxhlet extraction using Soxtec system 1046 (Tecator Ab, Hoganas, Sweden).

2.4. Bacterial challenge

Challenge test was performed following the method described by Choi et al. (2008). At the end of the feeding trial, fish were redistributed based on their earlier dietary treatments in 40-L tanks as triplicate groups of five fish and allowed to stabilize for 24 h. E. tarda (FPC 799) was grown in heart infusion broth (Nissui, Japan) for 24 h at 37°C. The culture broth was centrifuged at 3000× g for 10 min. Fish from each experimental group were injected intraperitoneally with 0.1 mL of the bacterial suspension per fish at a concentration of 1× 10⁸ colony forming units (cfu) mL⁻¹. Twenty-four hours after challenge, the flow rate was established (0.8 L/min) and mortality of the challenged fish from each tank was observed up to 12 days.

2.5. Statistical analysis

Data were subjected to one-way analysis of variance (ANOVA) test using Statistix 3.1 (Analytical Software, St Paul, MN, USA). When a significant treatment effect was observed, a least significant difference test was used to compare means. Treatment effects were considered significant at P < 0.05. The breakpoint for optimum dietary Macsumsuk^® was estimated by using the broken-line model of Robbins et al. (1979).

3. Results

3.1. Growth performance and haematological parameters

Table 3 shows the growth performance and haematological parameters of fish fed the different experimental diets. At the end of eight weeks of feeding trial, WG, SGR, FE and PER of fish fed M_1.2 diet were significantly higher than those fed M₀ diet (P < 0.05). However, there were no significant differences in WG, SGR, FE and PER among fish fed M_0.3, M_0.6, M_1.2 and M_2.4 diets, or among those fed M₀, M_0.3, M_0.6 and M_2.4 diets (P > 0.05), respectively. There was no significant difference in survival of fish fed all the experimental diets. RBC, Hb and HCT of fish fed M_0.6 diet were significantly higher than those fed M_2.4 diet. However, there were no significant differences in RBC, Hb and HCT among fish fed M₀, M_0.3, M_0.6 and M_1.2 diets, or among those fed M₀, M_0.3, M_1.2 and M_2.4 diets. Broken-line analysis of WG indicated that the optimum inclusion level of dietary Macsumsuk^® as an additive in juvenile Nile tilapia, O. niloticus (1.0–7.4 g) could be approximately 0.803% (Figure 1) under the given experimental conditions.

Figure 1. Broken-line regression analysis of weight gain (%) in juvenile Nile Tilapia, O. niloticus, fed five experimental diets.

Table 3. Growth performances and haematological parameters of juvenile Nile Tilapia, Oreochromis niloticus, fed five experimental diets.

	Diets
Parameters	M0	M0.3	M0.6	M1.2	M2.4	Pooled SEM
WG (%)	549.2^b	582.8^ab	598.2^ab	608.3^a	586.3^ab	11.7
SGR (% day^−1)	0.100^b	0.101^ab	0.101^ab	0.102^a	0.101^ab	0.004
FE (%)	79.5^b	84.0^ab	85.4^ab	86.4^a	82.8^ab	1.04
PER	3.34^b	3.53^ab	3.58^ab	3.70^a	3.56^ab	0.07
RBC (×10^6 cell/µg)	1.80^ab	1.81^ab	2.36^a	1.91^ab	1.51^b	0.12
Hb (%)	6.46^ab	6.70^ab	8.40^a	6.83^ab	5.30^b	0.43
HCT (%)	24.7^ab	24.2^ab	31.4^a	25.8^ab	21.0^b	1.47
Survival (%)	100	100	100	97.3	98.7	0.42

Note: Values are means from duplicate groups of fish where the values in each row with different superscripts are significantly different (P < 0.05).

WG: weight gain (%) = (final weight–initial weight) × 100/initial weight.

SGR: specific growth rate (%) = (loge final weight–loge initial weight)/day.

FE: feed efficiency (%) = wet weight gain (g) × 100/dry feed intake (g).

PER: protein efficiency ratio = wet weight gain/protein intake.

RBC: red blood cell (× 10⁶ cell/µg).

HCT: hematocrit (%).

Hb: Haemoglobin (%).

Pooled SEM: pooled standard error of means: SD/√n.

3.2. Whole-body proximate composition

Whole-body proximate composition of juvenile Nile tilapia fed different levels of dietary Macsumsuk^® is shown in Table 4. Whole-body crude lipid content was significantly higher in fish fed M_0.3 and M_0.6 diets than in those fed M_2.4 diet (P < 0.05). However, there were no significant differences in whole-body crude lipid content among fish fed M₀, M_0.3, M_0.6 and M_1.2 diets, or among those fed M₀, M_1.2 and M_2.4 diets (P > 0.05). Whole-body moisture content was significantly higher in fish fed M₀ diet than in those fed M_0.6 diet. However, there were no significant differences in whole-body moisture content among fish fed M₀, M_0.3, M_1.2 and M_2.4 diets, or among those fed M_0.3, M_0.6, M_1.2 and M_2.4 diets. There were no significant differences in whole-body crude protein and ash contents of fish among all treatments.

Table 4. Whole-body proximate composition of juvenile Nile Tilapia, Oreochromis niloticus, fed five experimental diets.

	Diets
	M0	M0.3	M0.6	M1.2	M2.4	Pooled SEM
Crude protein	16.41	16.32	16.72	16.41	16.82	0.12
Crude lipid	6.53^ab	7.56^a	7.62^a	6.81^ab	5.58^b	0.26
Crude ash	4.66	4.66	4.85	4.62	4.69	0.04
Moisture	72.08^a	71.37^ab	70.57^b	71.60^ab	71.46^ab	0.21

Note: Values are means from duplicate groups of fish where the values in each row with different superscripts are significantly different (P < 0.05).

Pooled SEM: Pooled standard error of means: SD/√n.

3.3. Challenge test

Figure 2 shows a cumulative mortality rate (%) of juvenile Nile tilapia over the duration of 12 days after intraperitoneal injection with E. tarda fed five different diets for eight weeks. Mortality was recorded right from the first day of the injection. After nine days of the injection, more than 40% of fish fed M₀, M_0.3, M_0.6, M_1.2 and M_2.4 diets had died. However, no significant differences were recorded in mortality rate of fish among all treatments (P > 0.05).

Figure 2. Cumulative mortality rate (%) after intraperitoneal injection with E. tarda in juvenile Nile Tilapia, O. niloticus, fed five experimental diets.

4. Discussion

To the best of our knowledge, no previous work has been done to investigate the effect of Macsumsuk^® on growth performance and feed utilization in fish. In the present study, fish fed diet containing 1.2% dietary Macsumsuk^® had a significantly higher growth performance and feed utilization efficiency as compared to fish fed the control diet. The present study demonstrates that the dietary supplementation of Macsumsuk^® enhanced WG, SGR, FE and protein efficiency ratio of Nile tilapia, as a feed additive. Likewise, Dada (2012) who reported that feeding Superliv® as a feed additive in Nile tilapia can enhance growth and feed utilization. In addition, Meurer et al. (2009) showed that the diet supplemented with brown propolis improved WG of fingerlings Nile tilapia, O. niloticus. These results may be possibly due to the Macsumsuk^® as a feed additive can enhance feed ingestion and absorption and nutrient metabolism in Nile tilapia; various types of feed additives enhance the digestibility and utilization efficiency of nutrients, including enzyme secretion; stimulators of in the digestive process by improving absorption, mobilization and transport of nutrients; and feeding stimulants that reduce feed/nutrient waste (Ceulemans et al. 2009).

Haematological parameters are used as valuable biological indicators in response to dietary manipulations (Adhikari et al. 2004; Shah & Altindag 2005; Maheswaran et al. 2008). In the present study, haematological parameters of Nile tilapia were taken into account and significant differences were found among different dietary treatments. Higher red blood cell (RBC), HCT and Hb values were found in fish fed Macsumsuk^® level of 0.6%. On the contrary, RBC, HCT and Hb decreased above this level, indicating that high inclusion levels of Macsumsuk^® above the optimum level negatively affect the haematological parameters of Nile tilapia. In other words, adequate supplementation of Macsumsuk^® improves growth, FE and haematological parameters of Nile tilapia; however, excess supplementation could adversely affect the health of fish. This suggests that care should be taken to maintain the optimum supplementation level. To our knowledge, there is no available information on the effects of dietary Macsumsuk^® on haematological parameters of fish species. Similarly, the obtained results in HCT concerning the effect of Macsumsuk^® are supported by the findings of some researchers such as Bae et al. (2012) who showed that HCT concentration of the juvenile eel, Anguilla japonica, decreased above the optimum level of dietary Propolis.

E. tarda, FPC 799 is a gram-negative bacterium associated with freshwater ecosystems. Known to colonize in a wide variety of amphibians, reptiles and fish, E. tarda can also cause disease in these animals, as exemplified by emphysematous putrefactive disease of catfish (Meyer & Bullock 1973). Edwardsiella septicaemia occurs in numerous freshwater and marine fish species economically important to aquaculture such as Nile tilapia, O. niloticus (Meyer & Bullock 1973; Austin & Austin 1999). The Nile tilapia is quite susceptible to E. tarda infection; consequently, in order to assess the disease tolerance of Nile tilapia in this trial, fish were infected with E. tarda. In the present study, there was no significant difference among different treatments in mortality of Nile tilapia infected with E. tarda. Therefore, Macsumsuk^® improved survival in Nile tilapia fed diets supplemented with 0.3–2.4% Macsumsuk^® nine days after intraperitoneal injection with the bacteria. It can be anticipated that dietary Macsumsuk^® was able to enhance the immune system in Nile tilapia against E. tarda infection.

In conclusion, results indicated the optimum Macsumsuk^® supplementation levels of 0.803–1.2% based on the broken-line analysis of WG and ANOVA test for optimum growth and FE and 0.3–2.4% for enhanced disease resistance in juvenile Nile tilapia, O. niloticus. This study may suggest that the dietary Macsumsuk^® can enhance growth performances, feed utilization, haematological parameters and induce higher protection of Nile tilapia against E. tarda infection.

Additional information

Funding

This work was supported by the Korea Institute of Industrial Technology and partially by the Feeds and Foods Nutrition Research Center (FFNRC). Macsumsuk^® was kindly provided by Macsumsuk GM (Co., Ltd.).