Abstract: Background and Objective: Phytobiotics as additives in aquaculture feed are increasingly recognized as feed for young and adult fish. Spirulina improves digestion and direct fats in the fish to growth. The beneficial effect of inclusion of Spirulina platensis as feed additives in tilapia diets during their development was studied to estimate its effect on tilapia reproductive performance. Materials and Methods: A phytobiotic Spirulina platensis was introduced to tilapia feed for 19 weeks at concentrations of (0.5, 7.5, 10 and 20 g kg–1 Spirulina platensis diet named: S0, S5, S7.5, S10 and S20, respectively). One hundred fish were divided to 5 groups and fed diets supplemented with algae at variable levels as well as control. The gonadosomatic index (GSI), fecundity, percentage of spawned eggs, egg and fry qualities were evaluated. Data were analyzed using one-way analysis of variance (ANOVA) at significance of (p<0.05). Results: Fish weight, GSI, spawning activity and spawned eggs number showed significant increase (p<0.05) on higher supplementation of phytobiotics and also fry length and weight increased in all the groups tested compared to control. Fry mortality and deformations percentage showed significant decrease (p<0.05) in phytobiotic fed fish. Group supplemented with algal level of (20 g kg–1 Spirulina platensis diet) in their feed spawned earlier at 45.3±7.8 days compared to 49.5, 51.3, 54.1 and 66.7 days for fish fed the 10, 7.5, 5 g kg–1 Spirulina platensis diet and control, respectively. Also, percentage of spawned eggs, total number of eggs/female recorded significant increase (p<0.05) in groups of fish supplied with the 20 and 10 g kg–1 Spirulina platensis diet. Eggs diameter, volume as well as weight showed insignificant change, while hatchability percentage increase in Spirulina fed fish compared to fish receiving control diet. Conclusion: Inclusion of Spirulina in fish feeds improve reproduction performance of tilapia fish, suggesting that Spirulina is an appropriate reproduction-stimulating additive in Nile tilapia culture.
INTRODUCTION
Tilapia (Oreochromis niloticus) is one of the most important economic freshwater fish of Egypt. Tilapia is considered suitable for culture because of their relatively fast growth, good utilization of artificial diets, high tolerance to adverse environmental conditions, high diseases resistance and easy breeding1.
The importance of phytobiotics as additives in aquaculture feed are increasingly recognized as a feed for young and adults fish. Spirulina improves digestion through production of extra cellular enzymes (protease and lipase) and able to direct fats in the fish for growth rather than storage2. Spirulina platensis, contain 60-70% by weight protein, vitamins B-12, carotene, minerals, essential amino acids (62%) and fatty acids3. Its lipid contents are similar to that of vegetable oils4, rich in linoleic 18:2n6 and α-linolenic 18:3n3 acids and their C20 derivatives5,6. Some of the cyanobacteria tend to contain large quantities of the total fatty acids, polyunsaturated fatty acids (PUFA) (20-60%), Eicosapentaenoic acids 20:5n3 and Arachidonic acids 20:4n6 that influence reproductive performance in fish7. Olvera-Novoa et al.8 indicated the usefulness of Spirulina for partial replacement upto 40% of the fish meal protein in Nile tilapia diets. El-Sheekh et al.9 suggests the possibility of using Spirulina as commercial nutrient source for large scale culture of fish in general and tilapia in particular. Takeuchi et al.10 and Elsayed et al.11 concluded that Spirulina is an appropriate growth-stimulating additive in tilapia culture. Lu et al.12 indicated that Spirulina is an effective uni-feed for larval tilapia at a feeding rate of 30% of body weight. Ibrahem et al.13 and Ungsethaphand et al.14 documented the use of Spirulina for enhancing growth parameters and disease resistance in fish. Research on the effect of feeding phytobiotics on the reproductive performance of tilapia fish is limited. Using Spirulina alga as the main feed to Nile tilapia increase, egg quality and survival rates, compared with standard fish feed15. Studies have reported the effect of Spirulina on fish reproduction. Spirulina enhanced seed production in yellow tail cichlid, Pseudotropheus acei 3, in goldfish, Carassius auratus16, swordtail2 and bassa fish, Pangasius bocourti17.
The present study aimed to discover the beneficial effect of Spirulina platensis as additive in diets of tilapia, Oreochromis niloticus on their reproductive performance and to assess possibility of using Spirulina as reproduction-stimulating.
MATERIALS AND METHODS
Fish breeding: The study was carried out at Aquaculture laboratory of the Arab Academy for Sciences and Technology, Alexandria, Egypt at the period from 15/3/2016-8/8/2016. The experiment was carried out using hundred Nile tilapia Oreochromis niloticus (body weight 55±2.5 g and total length 12.2±0.40 cm) which were obtained from the same parental breed stock pair held at Barseek fish farm at Behera Governorate and transported alive to the Aquaculture laboratory of the Arab Academy for Sciences and Technology, Alexandria, Egypt and left acclimated for 2 weeks prior to the experiments. Before the start of the experiment females were tagged using threading needle and a surgical thread. The females were pierced dorsally and a different colored tag was tied to identify females during spawning. They were divided into 5 equal groups each consisting of 2 replicates [10 fish/replicate (7♀, 3♂)]. Fish in each replicate were reared in aquaria (0.5 ×0.5×1 m, each) supplied with chlorine free tap water which was partially renewed daily.
They were fed twice daily on a basal diet (~34% protein and ~8.2% lipid) at 5% of body weight/day. Fish were weighed every 15 day and amounts of feed were adjusted according to the new weight. The water was cleaned regularly to remove unused feed and fecal materials and water were partially renewed daily. The temperature was maintained at 28±0.5°C using electric heaters, dissolved oxygen was measured using dissolved oxygen meter (Jenuay Model 9070 water proof meter) and continuous aeration was provided by air pumps. Dissolved oxygen ranged from 7-8 mg L–1 during experiments period.
Reproductive parameters: Reproductive performance of the females fed the different Spirulina platensis diets were observed all over the period of experiment (19 weeks). Females were observed daily to check whether it was about to spawn (with red and protruding genital papilla) or spawned. When eggs were seen in the mouth of females, all eggs in the buccal cavity were removed completely using a plastic dropper and the number of eggs from each female was counted. Eggs from females spawned naturally were used for periodicity (mean number of days taken for an individual female to complete one spawning cycle) and fecundity estimations. Post-spawning females were weighed. Absolute fecundity and relative fecundity were determined as mean number of freshly spawned eggs at each spawning/female or/gram body weight respectively, for determination of percentage of spawned eggs; after each spawning one freshly spawned female from each treatment was sacrificed and a segment of middle region of ovary (1 cm) was fixed, dehydrated, cleared, imbedded, sectioned, stained and microscopically examined. The number of post ovulatory follicle to total oocytes number was determined.
Eggs were artificially stripped from the ovarian cavity of females with red and protruding genital papilla by gentle pressing female belly. Ovulated eggs were fertilized by milt from males of same experimental group. Artificial spawns were used to determine eggs and fries quality. A subsample of 20 eggs from each replicate were measured (long, short axis) microscopically using eyepiece micrometer. Artificially fertilized eggs were placed to hatch in bottomless cylindrical container with a mesh at base supplied with a gentle flow of dechlorinated tap water to keep eggs rolling (mimic maternal incubation).
Two groups of fertilized eggs (20 eggs/treatment) were incubated till hatching to determined hatch ability rates, time to hatch and yolk-sac absorption, survival rate and the larvae length and weight. Unhatched eggs were counted. Deformity was determined as the ratio of normal larvae to larvae with curved body or tailless ones. At the end of experiment, fish were weighed, sacrificed and their ovaries weighed and their gonad index was calculated:
pirulina platensis: Spirulina platensis was obtained as a fresh powder reared in the Aquaculture Research Laboratory in Arab Academy for Science, Technology and Maritime Transport, Alexandria, Egypt.
Rations: A standard commercial ration formulated to fulfill dietary requirements of Nile tilapia were used containing crude protein, crude lipid, vitamins and minerals (Table 1). Five experimental rations were formulated containing feed additives for Nile tilapia: One as a control diet without supplements and a supplements at 5, 7.5, 10 and 20 g kg–1 Spirulina platensis diet where used. The basal and tested diets were formulated from the commercial feed ingredients. All the dry ingredients were grounded through a feed grinder. Experimental diets were formulated to be isocaloric (~8.2% lipid) and isonitrogenous (~34.5% protein) (Table 1). The ingredients were mixed with water in meat mincer with a 1mm diameter and the pellets were left to dry at room temperature. The required diet was prepared weekly and stored in plastic bags, kept in refrigerator at 4°C for daily use.
Statistical analysis: The data presented as Mean±SD, analyzed using one way analysis of variance ANOVA, followed by Duncan’s test using the SPSS package, version 17 (Chicago, IL,USA). Data were statistically significant at p<0.05. Homogeneity of data was confirmed before test.
RESULTS
Reproductive performance of fish
Spawning parameters: The reproductive parameters of female Nile tilapia fed the experimental feeds all over experiment period (19 week) is illustrated at Table 2. Fish weight decreased in fish feed Spirulina supplemented diets recording significant decrease (p<0.05) in males and females fish fed the S20 diet. No fish mortality was recorded in all tested groups during the period of experiment.
Tilapia fed on Spirulina supplemented diets showed better spawning performance compared to fish fed the controldiet (Table 2).
The percentage of spawned eggs increased (p<0.05) significantly in fish fed S20, S10 and S7.5 diets. The highest percentage of spawned eggs observed in fish fed the S20 diet (6.5±1.6) compared to (3.8±0.7) at S0 diet. Fish fed on Spirulina supplemented diet at level of 20 g kg–1 Spirulina platensis diet (S20) diet were the first to spawn for the first time (45.3 days) compared with 66.7 days in females fed the standard diet 0 g kg–1 Spirulina platensis diet (S0). Females fed S20 or S10 diets had the highest total eggs number (p<0.05) with respect to fish or tank (Table 2). No significant difference in absolute or relative fecundity values was recorded among fish fed the various diets. Fish fed the two diets S20 and S10 have the shortest inter-spawning interval that differ significantly (p<0.05) with fish fed the standard (S0) or Spirulina supplemented diet (S5).
Weight, diameter and volume of eggs from the four successive spawns did not show significant differences between fish fed the different Spirulina diets and control (Table 3). Fish fed the Spirulina diets have egg hatchability rates higher than eggs of fish fed the control diet (Table 4). Oreochromis niloticus females that fed Spirulina diets at all levels, showed significant increase in egg hatch ability (p<0.05). The incubation time of eggs to hatch (72-78 h) as well as the time for larval yolk sac to be absorbed (6-7days) did not differ among treatment at the fourth spawn. All Spirulina fed fish produced better larval quality with significant increases in fry weight and length (p<0.05). Percentage of dead fry significantly decreased in larvae of fish feed the spirulina supplemented diets compared to those fed the S0 diet (Table 4).
| Table 1: | Composition and proximate analysis of the experimental diets on dry weight basis |
Vitamins and minerals premix kg–1 diet:75000IU Vit. A: 9000IU Vit. D3: 150 mg Vit. E: 30 mg Vit. K3: 26.7 mg Vit. B1: 30 mg Vit. B2: 24.7 mg Vit. B6: 75 mg Vit. B12: 225 mg Nicotinic acid, 69 mg Pantothenic acid: 7.5 mg Folic acid: 150 mg Vit. C: 150 mg Biotein: 500 mg Choline chloride: 300 mg DL-methionine: 93 mg Fe: 11.25mg Cu: 210 mg Zn: 204 mg Mn: 5 mg Se and Co 5 mg. Diets analyzed according to AOAC18 | |
| Table 2: | Reproductive parameters of female tilapia fed Spirulina supplemented diets all over period of 19 weeks |
Mean±SD of two groups of four ♀ tilapia/treatment, different superscripts in same row were significantly different( p<0.05), 1( No. of eggs/fish/spawning), 2(No. of eggs g–1 b.wt/spawning) |
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| Table 3: | Mean eggs diameter, volume and weight from four consecutive spawns of♀tilapia fed Spirulina supplemented diets |
Mean±SD of two groups of ♀ tilapia/treatment, Egg diameter= (Long axis×short axis)/2 and Egg volume= π/6×(Long axis×short axis) | |
| Table 4: | Egg hatchability and larval quality from the fourth spawning of tilapia fed diets supplemented with Spirulina levels |
Mean±SD of two groups per treatment of ♀ tilapia fed diet supplemented with different levels of Spirulina. Different superscripts in same row were significantly different (p<0.05) |
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| Table 5: | Mean percentage(%) of spawning female tilapia over 4 successive spawns |
Mean±S.D. of two groups of four ♀ tilapia per treatment. Different superscripts in the same column were significantly different (p<0.05) | |
Also, percentage of deformed fry decreased significantly (p<0.05) in fish fed phytobiotic feeds with respect to fish fed standard diet, having maximum values (4 and 3% for dead and deformed fries) in fish fed the standard feed, minimum values (3 and 2% for dead and deformed fries) in fish fed the phytobiotic feed. Regardless of diet the number of spawning females showed decreased values by time. After four successive spawning, 53% of the brood fish fed the S10 diet were still fecund compared to only 30 and 38% in fish fed the S0 or S5 diets, respectively (Table 5).
DISCUSSION
The incorporation of Spirulina in tilapia feed favorably influenced, fecundity, GSI, fry production and survival. Spirulina contain significant quantities of protein, lipids and fatty acids which are the main constituents of egg yolk. Also, its essential fatty acids content provide energy for spawning activities. Dahlgren19 demonstrate that lipids, essential fatty acids and proteins of fish diet influenced rate of vitellogenesis, development and maturation of oocytes.
The improvement in reproduction efficiency of Spirulina feed fed fish reported in this work may bealso due to its content of micronutrients (B group vitamins), this agree with the finding of Coves et al.20 who stated the important of B vitamins supply in elevating reproductive performance of fish.
In present study the incorporation of Spirulina in tilapia feed, reduced fry mortality and deformity in Spirulina feed-fed fish compared to control ones. The reduction in numbers of dead and deformed fry in fish fed the phytobiotic diet may be due to the presence of B group vitamins specially (vitamin B1 and B12) in Spirulina. Wooster et al.21 demonstrated that the reduction in the mortality of Atlantic salmon friesis due to the beneficial effect of thiamin (vitamin B1). In the present study, the weight and length of produced fries were greater in the Spirulina-fed fish could be attributed to the fatty acids contain of Spirulina. Similarly Mazorra et al.22 concluded that, Spirulina inclusion in fish feed, enhance the spawning success and quality of fries produced due to the lipid content of Spirulina specially n-6 fatty acids. Santiago and Reyes23also stated the important of n-6 fatty acids group specially ARA acid in improving spawning efficiency and fry morphometric characteristics. Through the experimental period, fish fed the Spirulina supplemented diets produced, greater number of eggs compared to fish feed on standard diet which was therefore due to increased percentage of spawned eggs, shorter inters pawning intervals, rather than absolute and relative fish fecundity, that did not showed significant changes among all treatments used. The supplementation of, Spirulina to fish feed in this study lead to a higher percentage of spawned eggs, higher hatch ability rate and fries survival percentage than those fed on control feed. Similarly Guroy et al.3, James et al.24, Vasudhevan and James16, James et al.2 and Meng-Umphan17 have reported that, Spirulina enhanced seed production in yellow tail cichlid, Pseudotropheus acei, in goldfish, Carassius auratus, in swordtail, Xiphophorus helleri and bassa fish, Pangasius bocourti respectively. Although there were no detected differences in egg morphometric characters, the quality (length and weight) of fries from fish fed on the Spirulina supplemented diets was higher compared to control that may be attributed to high Spirulina content of essential amino acids, fatty acids and vitamins that influence the growth performance of fish. Growth of the red swordtail, Xiphophorus helleri and gold fish was enhanced on addition of Spirulina at level of 8% in fish feed2,24.
CONCLUSION
Incorporation of Spirulina in diets of tilapia during their gonadal development improve reproduction performance of tilapia fish leading to higher fecundity and GSI values, better quality of eggs and larvae. Eliciting higher egg hatchability and larval survival, suggesting that Spirulina is an appropriate reproduction-stimulating additive in Nile tilapia.
SIGNIFICANCE STATEMENTS
This study discovers the possibility of improving fish breeding via studying the impact of Spirulina in fish feed on reproductive parameters of Nile tilapia. This study will help the researcher to uncover the critical areas of possibility of using Spirulina as reproduction-stimulating additive in Nile tilapia feeds.
ACKNOWLEDGMENTS
Authors are indebted to Assistant Associate Prof. Essam Abdel maula, Head of Aquaculture Research Lab. Arab Academy for Science and Technology, Alexandria, Egypt for supporting the present study and supplying the present study with Spirulina.