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This study was carried out to evaluate the effect of synbiotic (Biomin imbo) on growth performance, survival rate and reproductive parameters of Angelfish (pterophyllum scalare) via supplementation with Biomar. Four level of Biomar experimental diets (54% protein and 14% lipid) were prepared by adding synbiotic (0.15, 0.5, 0.75, 1 g/Kg) at the basal diet (Biomar) and the Angelfish larvae in experimental treatments were fed of the four levels of synbiotic with 5 percent body weight (3 times a day). The larvae in control treatment were fed without supplemented Biomar. The results showed that larvae fed the synbiotic had significantly increased final body weight in comparison to control treatment. After 90 days, 5 Adult female and male Angelfish were divided from each treatment. The results showed that fecundity in experimental treatments increased in comparison to control treatment. The synbiotic also had positive effects on hatching rate in comparison to those in control treatment but, there were no significant difference (P>0.05) among treatments however they were better in T3. The synbiotic also had significant positive effects on specific growth rate (SGR) and food conversation efficiency (FCE) in comparison to those fed the control treatment however in treatment T3 was more than T4 (T3>T4>T2>T1>control treatment).
Keyword: Angelfish, Pterophyllum scalare, Suplementation, Biomar, Growth Performance, Survival Rate, Female, Hatching Rate, Fecundity
The Angelfish (P. scalare) is one of the most popular aquarium species, as this species commands a higher price compared with most freshwater food species and other ornamental fish. In spite of the importance of Angelfish in ornamental fish culture, there has been neither research nor development of cost-effective feed for the intensive culture of this species. All ornamental fish feeds are 10-60 times higher in price than aquaculture feeds for food species. Second, the price of the feed targeted for a single ornamental species very dramatically compared to the price of food fish feeds, each of which is targeted for a specific species (Tamaru and Ako, 2000). For this reason, formulation feed rations for ornamental fish carry importance for aquaria sector (Sales and Janssens, 2003).
Synbiotics refer to nutritional supplements combining probiotics and prebiotics in a form of synergism, hence synbiotics, enhancing their isolated beneficial effects. When two nutritional ingredients or supplements are given together; the resulting positive effect generally follows one of three patterns: additivity, synergism or potentiation. Additive effect occurs when the effect of two ingredients used together approximates to the sum of the individual ingredient effects. In case of synergism, it is said to occur when the combined effect of the two products is significantly greater than the sum of the effects of each agent administered alone. The term potentiation is used differently, some pharmacologists use potentiation interchangeably with synergism to describe a greater than additive effect and others use it to describe the effect that is only present when two compounds are concurrently (Chou et al. 1991).
Synbiotics affects the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract by selectively stimulating the growth and/or by activating the metabolism of one or a limited number of health promoting bacteria, and thus improving the host "welfare". In humans, probiotics are mainly active in the small intestine while prebiotics are only effective in the large intestine, so the combination of the two may give a synergistic effect (Gibson and Roberfroid, 1995). The first application of synbiotics in fish is that of Rodriguez-Estrada et al., (2009).
The appropriate use of probiotics in the aquaculture industry were shown to improve intestinal microbial balance, and also to improve feed absorption, thus leading to increased growth rate (Fuller, 1989; Rengpipat et al. 1998), and also reduced feed conversion ratio (FCR) during the cultural period (Wang et al. 2005). Probiotics in aquaculture have been shown to have several modes of action: competitive exclusion of pathenogenic bacteria through the production of inhibitory compounds; improvement of water quality; enhancement of immune response of host species and enhancement of nutrition of host species through the production of supplemental digestive enzymes (Verschuere et al. 2009). Because Bacillus bacteria secrete many exoenzymes (Moriarty, 1998), these bacteria have been used widely as putative probiotics.
In Angelfish, as in all vertebrates, reproduction is regulated by the hypothalamus-pituitary-gonadal axis. The hypothalamus, integrating internal and external stimuli, releases Gonadotropin-releasing hormone (GnRH) (Zohar, et al, 2010). In recent years, it has been established that GnRH transcription and secretion are gated by the state of energy reserves in the organism (Hill et al, 2008). The impact of energy status on the reproductive axis is conveyed through a number of neuropeptide hormones and metabolic signals, such as kiss1, kiss2, and leptin, whose nature and mechanisms of action have begun to be deciphered only in recent years in mammals and, to a lesser extent, in fish (Casanueva and Dieguez, 1999). Under the influence of GnRH, the pituitary secretes follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which act upon the gonads controlling follicle growth and maturation (Patino et al. 2001). In particular at ovarian level, LH, through its receptor (LHcgr), stimulates the production of 17a-hydroxyprogesterone that is converted (by the action of cbr1l) into 17a, 20b-dihydroxy- 4-pregnen-3-one, the maturation-inducing hormone (MIH) (Patino et al. 2001). The binding of MIH to its receptors (paqr7b and paqr8) activates the maturation processes (Hanna and Zhu, 2009).
This study investigated for the first time the effects of synbiotic (Biomin imbo) on growth performance, survival rate, fecundity and reproductive factors in female' angelfish via supplementation with Biomar.
MATERIALS AND METHODS
Fish: Larvae of angelfish with initial weight, 0.57Â±0.1 g were obtained from an Institute of commercial supplier the Ornamental Fish Hatchery in Gorgan, Iran.
They were kept in glass aquaria (each with a dimension of 30Ã-40Ã-60 cm). This experiment was conducted in a completely randomized design with five treatments (four synbiotic levels and a control), and three replicates per treatment for a total of fifteen Larvae of angelfish. The density of fish larvae per aquarium were 5 fish. At the end of rearing, adult female and male zebra fish were divided from each treatment. The animals were kept in 50 L glass tanks under controlled conditions (28Â±0.5 Â°C and 14 h light: 10 h darkness).
Experimental diet: The synbiotic (Biomin imbo) was prepared from the commercial product Protexin aquatic (Iran-Nikotak). Also Biomar was provided by aquatic foods company. Four level of Biomar experimental diets (54% protein and 14% lipid) were prepared by adding synbiotic (0.15, 0.5, 0.75, 1 g/Kg) at the basal diet (Biomar) and the angelfish larvae in experimental treatments were fed of the four levels of synbiotic with 5 percent body weight for 3 times a day (6.00, 14.00 and 22.00). The control treatment was fed without supplemented Biomar.
Feed analysis: Nutrient compositions of experimental diets (Biomar) are given in Table 1. Proximate composition of diets was carried out using the Association of Analytical Chemists (AOAC, 2000) methods. Protein was determined by measuring nitrogen (NÃ-6.25) using the Kjeldahl method; Crude fat was determined using petroleum ether (40-60 Bp) extraction method with Soxhlet apparatus and ash by combustion at 550 Â°C.
Determination of growth parameters: growth parameters were calculated as follows: final body weight (WG) = final body weight (g) - initial body weight (g). Specific growth rate (SGR) (% BW day-1) = (Ln final body weight (g)-Ln initial weight (g))/(experimental period) Ã- 100. Feed conversion ratio (FCR) (%) = (total fed/body weight increase (g)) Ã- 100. Daily growth rate (DGR) = (final body weight (g) - initial weight (g)) Ã- (100)/(experimental periodÃ-initial weight (g)).
Determination of reproductive parameters: reproductive performances were calculated as follows: relative fecundity = Total larvae production at throughout experimental period/mean weight of female (g). Total larvae production per female = Total harvested throughout experimental period per number of female. Gonadosomatic index (%) = (Ovary weight/body weight) Ã- 100. Survival (%) = (Total live fish after production / initial fish throughout experimental period) Ã- 100 where it is the day of experiment.
Reproductive parameters: reproductive parameters were investigated for one pair of each replication. Feeding was continued during the spawning period. Spawning, hatching and survival for each pair of fish were investigated.
Statistical analysis: in order to determine significant differences, results were analyzed by one-way Analysis of variance (ANOVA) and Duncan's multiple range tests were used to analyze the significance of the difference among the means of treatments by using the SPSS program.
RESULTS AND DISCUSSION
Synbiotic effects on angelfish growth performance and survival rate:
The results of feeding, growth performance and survival rate presented in Table 2 and it clearly showed that the synbiotic had beneficial effects on the growth performance on Angelfish larvae. That larvae fed the synbiotic had significantly increased on growth performance in comparison to control treatment (P<0.05). Also, the four different treatments of synbiotic were significantly different for any of growth parameters. The greatest effect appeared to be obtained in treatments T3 (supplemented with 0.75 g/kg) and Angelfish larvae in treatment T3 clearly showed better growth in all of the growth performance than treatment T4. However, concentration of synbiotic in treatment T4 was more than treatment T3 but result was opposed (T3>T4>T2>T1>control treatment). This is particularly true for specific growth rate, where the highest was obtained in the experimental treatment T3. The food conversion ratio (FCR) in the experimental treatments was significantly decreased in comparison with control treatment (p<0.05). Effects of commercial probiotic on aquaculture has been investigated by researchers, and some of this research has not shown any positive effects on growth parameters or survival rate or any promising result on the cultural condition. For instance, Shariff et al. (2001) found that treatment of Penaeus monodon with a commercial Bacillus probiotic did not significantly increase survival or El- Dakar et al. (2007) found that treatment of Siganus rivulatus with commercial probiotic/prebiotic did not significantly increase survival rate but it had positive effect on growth performance. These results disagree with our findings, although fish and crustaceans may respond differently to probiotics.
The maximum of final body weight (FBW) were obtained in treatment T3 and T4 and were 15.87Â±0.15g and 14.17Â±0.06g respectively. Also, specific growth rate (SGR) for these treatments were 1.48Â±0.01% and 1.35Â±0.01% body weight/day respectively. The maximum of Feed Conversion efficiency (FCE) (0.94Â±0.0%) was observed in treatment T3. The lowest of growth parameters were obtained in control treatment, while the highest food conversion ratio (FCR) (1.3Â±0.1), were obtained in this treatment where the fish larvae fed by unsupplemented Biomar. Also supplemented Biomar with synbiotic had positive effect on survival rate. But, there were no significant differences (p>0.05) among treatments however they were better in T3. The lowest survival rate observed in control treatment (92.23Â±5.71) and had no significantly different to other treatments.
Gomez-gill et al. (2000) found selection of probiotic bacteria had beneficial effects for larva aquatic organisms. Bagheri et al. (2008) found that supplementation of trout starter diet with the proper density of commercial bacillus probiotic could be beneficial for growth and survival of rainbow trout fry. These findings agree with our results. Ghosh et al. (2003) indicated that the B. circulans, B. subtilis and Bacillus pamilus, isolated from the gut of Rohu, have extracellular protease, amylase, and cellulose and play an important role in the nutrition of Rohu fingerlings.
Synbiotic effects on angelfish reproductive parameters:
Reproductive parameters were investigated for each treatment in spawning (Table 3). Result showed that, there were no significant difference among treatments in fecundity and percentage of hatching.
Also, the four different treatments of synbiotic were not significantly different for any of reproductive indices that, among the three different concentrations of synbiotic supplemented with Biomar fed to angelfish, the greatest effect appeared to be obtained in T3 experimental treatments.
Reproduction is gated by the state of body energy reserves and is sensitive to different metabolic cues; the neuroendocrine mechanisms responsible for the tight coupling between energy homeostasis and fertility are represented by metabolic hormones and neuropeptides that integrate the hypothalamic center governing reproduction, controlling the expression and release of GnRH (Zohar et al., 2010; Castellano et al., 2009: Kitahashi et al., 2004).
Thus, full activation of the hypothalamic-pituitary-gonadal axis at puberty and its proper functioning in adulthood critically depend on adequate body energy stores (Hill et al., 2008). The identification of the adipose hormone leptin, which signals the magnitude of energy stores to the hypothalamic centers governing reproduction (Casanueva and Dieguez, 1999; Goumenou et al., 2003), represented an important step toward understanding the mechanisms controlling this interplay.