PHYTOGENIC FEED ADDITIVE (PFA) IMPROVED SURVIVAL AND GROWTH OF NILE TILAPIA (OREOCHROMIS NILOTICUS) FRY DURING SUBSEQUENT NURSING RAM C. BHUJEL1*, LUMPAN POOLSAWAT1 AND RUI A. GONÇALVES2 1
Aqua-Centre, Asian Institute of Technology (AIT), Thailand
2
BIOMIN Holding GmbH, Erber Campus 1 3131 Getzersdorf, Austria
ABSTRACT A two-stage trial was conducted to examine the efficacy of an experimental phytogenic feed additive (PFA) on Nile tilapia fry. Four doses of the phytogenic products 0 (control), 0.5 (low), 1.0 (medium) and 1.5g/kg feed (high) were tested in triplicate. A total of 1,200 fry (6.4±5.8mg) were stocked in 12 aquaria each and fed five times a day for 21 days. The fry were nurse further in aquaria and hapas-pond for another 21 days. Randomly sampled 300 fry from each aquarium were transferred to each of the 12 hapas. Another 300 fry from the same aquaria were transferred to clear water plastic tanks. During the 21-day feed treatment, fry survival was not affected by PFA. However, during subsequent nursing in hapa-in-pond system, 7% higher fry survival was found at 1.0 and 1.5 mg/kg diet compared with control, and in aquaria, 1.27mg/kg diet of PFA dose was determined for highest survival. Keywords: Nile tilapia, fry survival, Trichodina, parasite, phytogenic feed additive
INTRODUCTION Nile tilapia (Oreochromis niloticus) has become the most widely cultured freshwater species of fish with an annual global production of over 5 million tons in 2015 (Bhujel, 2014; Fitzsimmons, 2015). Recent technological advances, e.g. the commercial scale production of high quality monosex fry, have accelerated the growth of the industry (Bhujel, 2014). Annual fry production has reached over a billion in Bangladesh, Thailand and other countries (Bhujel, 2011). Tilapia fry production has become a good business, which is moving from traditional extensive to intensive systems (Bhujel, 2008; 2014). Fry are produced in specialized hatcheries and nursing of fry is done in 2 or 3 stages with varying periods of 1-2 months each. High quality large-size fingerlings J. Aqua Trop. Vol. 32, No. (3-4) 2017, Pages 427-439 © Prints Publications Pvt. Ltd. Corresponding Author E-mail:
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are always in high demand for grow-out culture. As farmers move towards high-density culture, the occurrence of disease is increasing. Consequently, special care is needed during the nursing period of fry and fingerlings. Survival of fry often drops to critically low i.e. 15-20%, during sexreversal and nursing. Infections by bacteria and viruses cause low survival; these are often secondary in nature i.e. after infection by various internal and external parasites. An effective approach to control parasites and improve survival is therefore necessary. Trichodina is the most prevalent parasite in Nile tilapia (Bhujel, 2014). According to Hassan (1999), ideal conditions for the occurrence of Trichodina are poor water quality such as low dissolved oxygen, high ammonia, high stocking densities, rough handling of fish and high loads of organic matter. Clinical signs of Trichodina sp., infection (also called Trichodinosis) are abnormal coloration, sluggish movement, appetite loss, weight loss, skin lesions, fin erosion, excess mucus production and gill necrosis. Trichodina is a bell shaped ciliate protozoa, which multiplies by binary fission. It has a sucking disc from a lateral view, whereas the dorsal view looks round. Formalin baths (final concentrations around 250 – 300 ppm) are the most effective way to control this parasite, but it is time consuming, leaves toxic residues in fish, decreases dissolved oxygen in water and it is not environment friendly (Hassan, 1999; Chitmanat et. al., 2005; Chitmanat, 2013). In larval rearing tanks, increasing the salinity to 5 ppt can reduce the Trichodina load especially during winter. Indeed, if nursing is done in coastal areas, where salinities are >5 ppt, Trichodina problems may be avoided. However, it is not feasible to increase the salinity of a whole pond. Furthermore, farmers do not favor this method as it might create problems in surrounding freshwater environments. Deen and Mohamed (2009) demonstrated that crude extracts of garlic (Allium sativum) and mugwort (Artemisia vulgaris) at 800 mg/L could control Trichodina sp., as well as Aeromonas hydrophila in Nile tilapia. According to Chimanat (2005), 800 mg/L of garlic extract and Indian almond (Terminalia catappa) could also reduce Trichodina infection in Nile tilapia fingerlings. Pandey (2013) proposed that phytogenic compounds could stimulate the non-specific immune system, which could explain this increased survival. Musman et al., (2015) also studied the efficiency of tannins (hydrolysable tannins, condensed tannins and mixed tannins) extracted from the leaves of Avicennia marina on Trichodinosis inhibition. They found that condensed tannins were the best type for the control of ecto-parasites. However, tannins are astringents, plant poly-phenolic compounds that bind to and precipitate proteins and various other organic compounds including amino acids and alkaloids. This means they have anti-nutritional properties that could inhibit growth. An alternative solution to control Trichodina is needed. Phytogenic feed additives (PFA’s) could be one solution. Phytogenic compounds compose of a wide variety of herbs, spices, and products extracted mainly from plants and are primarily essential oils. They have many favorable characteristics including improving the palatability of feed, have anti-oxidative and antimicrobial properties and enhance activities of digestive enzymes and nutrient absorption by enhancing Journal of Aquaculture in the Tropics
Phytogenic Feed Additive (PFA) Improved Survival and Growth of
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intestinal secretions (Windisch et al., 2008). Phytogenic compounds are also believed to improve feed utilization, growth performance and immune system in aquatic animals (Gonçalves and Santos, 2015). The use of phytogenic compounds to control parasites has already been attempted via dipping with limited success. Recently, diverse natural extracts are produced and being tested as feed additives. Menanteau-Ledouble et al., (2015) used a feed additive application, demonstrating enhanced survival in rainbow trout (Onchorhynchus mykiss) when fish were infected by Aeromonas salmonicida. Similarly Dada (2015) tried Fluted Pumpkin (Telfairia occidentalis) leaf powder as feed additive in African Catfish (Clarias gariepinus) fingerlings. However, no studies have been tried in Nile tilapia. The present study therefore, was carried out to evaluate the efficacy of a PFA on survival and growth performance of Nile tilapia fry.
MATERIALS AND METHODS Experimental site and units The experiment was conducted at the Asian Institute of Technology (AIT), Thailand. A total of 14,400 Nile tilapia (Oreochromis niloticus) fry were obtained from the AIT tilapia hatchery and 1,200 fry (average weight of 6.4 ± 5.8 mg) were acclimated in each of the 12 aquaria (60 cm x 40 cm x 40 cm) for 7 days. After that they were fed with experimental diets for 21 days (n = 3). Then, the fry were separated into two groups (3,600 fish/group). The first set of treatment groups (n = 3) were transferred to 12 nursing hapas (50 cm x 30 cm x 80 cm) installed in a pond (400 m2) where parasites were found. The second set of all the treatment groups were kept in the same 12 aquaria (60 cm x 40 cm x 40 cm) with clear water controlled-environment without any parasites. Both the groups were nursed for 21 days. Detailed schematic diagram has been given in Figure 1.
Figure 1. Experimental design and feeding regime during sex-reversal with hormone feeding and parasite challenge phase during nursing phase with commonly used feed (Table 1 & 2). Journal of Aquaculture in the Tropics
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Feed and experimental design A phytogenic compound (BIOMIN Holding GmbH, Austria) was incorporated in standard sexreversal diets at four dosages; 0 (Control; C), 0.5 (Low; L), 1.0 (Medium; M) and 1.5 (High; H) g per kg of feed (Table 1). The supplemented diet was fed to the tilapia fry for 21 days five times daily. The fry were fed 5 times a day at 08:00, 10:00, 12:00, 14:00 and 16:00 hrs during acclimation and sex-reversal. Following the first phase, each group was split into two for nursing; one set of fry were raised in clear water system (without parasites) with the group C, L M, and H, and another half were raised in an open pond where Trichodina sp. was found. During this nursing period, they were fed twice daily (09:30 and 15.30 hrs). The treatments notations were CP, LP, MP and HP, where P stands for parasites. The feed formula and the composition of the two diets are shown in Tables 1 & 2. Table 1: Feed formulation and proximate composition of sex-reversal and common diets Standard diet for sex-reversal
Common diet for fry nursing
Ingredients
Composition (g)
%
Composition (g)
%
Fish meal
1,000
98.03
500
49.02
Rice bran
0
0.00
500
49.02
Aqua-mix
20
1.96
20
1.96
17-MT
0.06
0.01
0
0.00
Total (g)
1,020.06
100
1,020
100
Table 2: Feed formulation and proximate composition of sex-reversal and common diets Diet for sex-reversal
Common diet for fry nursing
Dry matter (%)
93.2
91.7
Moisture (%)
6.8
8.3 Proximate composition (% DM basis)
Crude protein
63.47
41.44
Crude lipid
11.98
12.44
Crude fiber
0.25
3.82
Nitrogen free extract
6.93
28.56
Ash
17.37
13.74
Total
100.00
100.00
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Phytogenic Feed Additive (PFA) Improved Survival and Growth of Nile Tilapia (Oreochromis niloticus) Fry during Subsequent Nursing
Analytical method Fish survival, final weight and specific growth rate (SGR) were calculated at the end of each trial. Three samples of whole body mucus, caudal fin and gill arches were removed every three days for the examination presence of ecto-parasites. These data were compiled in MS Excel and analyzed using SPSS applying one-way ANOVA and Student’s t-test and regression considering the significance level of 5%. Water quality During the 21-day feeding trial, dissolved oxygen (DO), temperature and pH were monitored every day at 07:30 hrs by using DO meter (Cyberscan DO 110 with RS232, EUtech, Instrument), and ammonia (NH3) was analyzed every 4 days at 07:30 hrs. Water in aquaria was changed approximately 70% daily in the evening during acclimation and sex-reversal treatment period. In addition, DO, temperature, pH and NH3 were recorded every 7 days at 07:30 hrs during nursing. Water in second nursing tanks was changed approx. 50% every 2 days in the evening. No water was exchanged in the nursing pond.
RESULTS Trial 1 (Sex-reversal period) Results showed that average SGR during the acclimatization (7 days) and sex-reversal period (first 21 days) ranged from 10.6 – 11.2% and the survival ranged from 63.5 – 65.8% (Table 3). However, the SGR and the survival of fry were not significantly (P>0.05) different among all the doses of the PFA. There is a decreasing trend of final and mean weights of fry with the increased level of PFA. Similarly, with the increasing dose of PFA dose, there were a general trend of increasing FCR and decreasing protein efficiency ratio (PER) was found. Table 3: Growth and survival of fry during sex-reversal period supplemented with PFA Initial Weight
Final Weight (g)
SGR (% /day)
Survival of fry (%)
(g)
Mean
SD
Mean
SD
Average
SD
0 (Control)
0.006
0.154 a
0.006
11.2 a
0.13
64.2 a
2.5
0.5 g/kg (Low)
0.006
0.144 a
0.016
11.0 a
0.39
65.1 a
2.7
1.0 g/kg (Medium)
0.006
0.132 a
0.014
10.6 a
0.38
65.8 a
2.4
1.5 g/kg (High)
0.006
0.131 a
0.010
10.6 a
0.28
63.5 a
3.0
Note: Means with same superscripts are not statistically different at 5% level of significance. Journal of Aquaculture in the Tropics
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INSERT TABLE 3 Trial 2 (Subsequent nursing for 21 days) During the nursing period of 21 days, final weight ranged from 0.510 – 0.590 g, SGR from 5.18 – 7.43 % and survival ranged from 55.3 – 63.5% (Table 4). Interestingly, phytogenic feed additive has significant (P
