The unstable production of grouper fry in the hatchery is one of the constraints in the

Effect of KIKO technology on growth and survival of grouper Epinephelus fuscoguttatus larvae Ofelia S. Reyes Aquaculture Department Southeast Asian Fisheries Development Center 5021 Tigbauan, Iloilo, Philippines Introduction The unstable production of grouper fry in the hatchery is one of the constraints in the development of grouper industry globally. Several breakthroughs in grouper larviculture have been reported to address the problems of inconsistent supply of grouper fry. To date however survival of grouper fry from the hatcheries is still low compared with other marine species. Several studies were carried out to improve the survival of grouper fry in the hatchery. Nutritional content and size of live food fed at the early stage of grouper (Duray, 1994), application of thyroid hormones to accelerate metamorphosis (De Jesus et al., 1998), stocking density (Duray et al., 1997), size and background colour of larval tank (Duray et al., 1996) were among the studies done under hatchery condition at SEAFDEC/AQD. Other limiting factor that affects the fry production of grouper in the hatchery is the water quality. Water quality directly affects feed efficiency, growth rates, survival, and health of the fish. High mortality, disease outbreak, poor growth, poor food

utilization and abnormalities are directly related to poor quality of the larval rearing water. Water temperature, salinity, oxygen, ph, ammonia and nitrite are key water parameters that have to be managed in the hatchery. Management of these parameters will provide a relatively stress free environment to meet the physical, chemical and biological standards for normal growth and survival of larvae under hatchery condition. Ammonia is the principal nitrogenous end product excreted by the fish while nitrite is an intermediate in the bacterial nitrification of ammonia and can build up in biological filters of the water recirculating systems. The toxicity of the aqueous ammonia solution is directly related to the amount of un-ionized ammonia (NH 3 ) present. The proportion of NH 3 to NH 4 increases with increasing ph and temperature (Trussell, 1972). Other environmental factors affecting the toxicity of ammonia include low concentration of oxygen, carbon dioxide and salinities above and below blood isotonicity (EIFAC, 1973). Low nitrite levels can be toxic to fish however increase in dissolved chloride and calcium or ph, decreases or inhibits the toxicity of nitrite to the fish (Perrone & Meade 1977; Russo et al 1981). Some species of marine fish larvae are affected when the culture water has higher concentration of ammonia. The newly hatched larvae were observed to have lower tolerance to ammonia concentration compared to older larvae. Objectives

A study was conducted to determine the effect of Kiko Technology on the growth, and survival of grouper fry. The specific objectives of the study were: 1. To determine if the Kiko Technology can improve the water quality, specifically ammonia and nitrite in the larvae culture of grouper, Epinephelus fuscoguttatus. 2. To determine if survival and growth of grouper fry can be improved using the Kiko Technology METHODOLOGY Newly-hatched grouper larvae were used in the experiment. Treatments tested were as follows: Treatment 1 no Kiko as control; Treatment 2 with Kiko. Two runs (Sept. 6.-Oct. 4, 20113 and Nov.-Dec. 2013) were done using 3-ton circular tanks with three replicates for each treatment. Another run (Feb. 8- March 21 2014) was conducted using 5-ton circular tanks with four replicates for each treatment. This experiment was done to determine the effect of temperature in the culture system. Sampling was done at 10, 15, 25, 35 and 45 dph to monitor the growth and survival of fry. Furthermore the culture period was extended until day 45 when dorsal and ventral fins of the fry were resorbed. A complete randomized design was followed. Grouper eggs were collected from the Big Hatchery Complex at SEAFDEC/AQD and transported to finfish hatchery for incubation and hatching. Eggs were treated with iodine solution before incubation at 50 ppm to reduce the bacterial population and to avoid the occurrence of parasite infestation from the broodstock tank to the larval

rearing tanks. Newly hatch larvae were stocked in circular tanks at stocking density of 15 larvae L-1. Initial body measurements were done from live images of fifty larvae using the microscope (national DC3-420T digital microscope NTSC system, 4X) where images were captured and stored in a computer. For weight measurement, digital balance (Symmetry PA220, Cole-Parme) was used. Ten E. fuscoguttatus fry were sampled for growth (length and weight) at 10 days post hatched (dph), 15 dph and 25 dph. After each sampling period, survival was estimated using the water column sampler. Three areas in the rearing tank were sampled. Water volume was determined and larvae were counted to obtain the number of larvae L -1. All fry from each tank were counted at the end of the experimental run. Larvae were fed enriched rotifer from day 2 to 15 at 3-15 rotifers/ml/day. Daily monitoring (am and pm) of rotifer and Artemia density was done to maintain the desired feeding level. Newly hatched Artemia was introduced in the rearing tank starting day 15 daily at 0.5 Artemia nauplii/ml. Enriched three day old Artemia were fed at 1 ind./ml daily starting 25 dph. Feeding ration of the Artemia biomass was adjusted if the feeding ration dropped below the desired ration. Static culture system was followed starting day 1 until day 30. Water temperature and salinity was monitored daily. Dissolved Oxygen and ph were monitored twice a week using YSI multi parameters. If dissolved oxygen levels decreased below 3 ppm, titration method was done to compare the dissolved oxygen reading of YSI with the

titration method. Ammonia-nitrogen and nitrite-nitrogen were determined using Phenate and colorimetric method. Results and Discussion In the preliminary run, survival and growth of grouper larvae reared with Kiko in tanks were 0.79% and 12.4 mg, while those in the control were 0.40% and 10.6 mg, respectively (Table 1). The first run after 28 days of culture, the weight of E. fuscoguttatus larvae reared in tanks with Kiko was slightly lower (11.7 mg) than the control group (13.60). No significant difference was noted in the body length in both treatments (Table 1). The percent survival was better in the control group (0.91%) compared with the group reared with Kiko in tanks (0.36%). In another run, mean final body weight of grouper larvae reared with Kiko Tech. was 6.75 mg while the control group had final body weight of 7.07mg. The total body length of the control group was 7.64mm and the fry reared with Kiko tech had a total body length of 7.47mm. The survival of larvae reared with Kiko and those in the control were 0.04% and 0.06% respectively after 30 days (Table 1). The survival rates of both treatments were very low, moreover the size of the fry were small compared to the previous runs. This low survival rate and small in size of grouper fry in both treatments could be due to very low temperature (22-24 o C) of the rearing water during the conduct of the experiment. According to Brownell et al. (1980) larvae at low temperature are inactive and do not feed. The same condition was observed in this

experiment. In addition it was observed during the conduct of the experiment, larvae stayed at the bottom of the rearing water. Hence, encounter of natural food was limited since rotifers stayed in the water column. Another run was conducted to determine the effect of temperature and the culture period. The final weight of the grouper fry reared in tanks with Kiko was significantly high (73.73 mg) compared to those fry in the control (54.53 mg). Slightly longer total body length was noted in the fry reared in tanks with Kiko was better than control. Furthermore, the colour of fry from tanks with Kiko was darker and size disparity among groups was minimal compared to those in the control. No significant difference was observed in the survival rate in both treatments (Table 2). The total ammonia-nitrogen was slightly elevated (1.52 ppm) during the first few days in tanks with Kiko treatment compared to the control, 0.83 ppm (Table 4) however towards the end of larval rearing, level of ammonia-nitrogen decreased to 0.22ppm. The nitrite-nitrogen content in both treatments (preliminary run) were elevated, see table 4. In the 1st run, the ammonia-nitrogen and nitrite-nitrogen level of the rearing water with Kiko Tech was lower compare to the control group. Only 1 observation noted with high nitrite-nitrogen level (0.35ppm). In 2 nd run, both the ammonia-nitrogen and nitrite-nitrogen level of the Kiko Tech treated water was slightly higher than the control, Table 4.

No significant difference observed in dissolved oxygen (DO), salinity, temperature, ph in both treatments of all the experimental runs conducted. No significant differences were noted in growth and survival of grouper fry for both treatments in all experimental runs done could be the too short (30days) duration of the experiments. Based from 3 rd run, better results in growth was determined when larval rearing period was extended until end of the metamorphosis of grouper fry. The water parameters determined during the larval rearing, no significant difference in both treatments was observed in all experiments done. References: Boyd, C.E.1990. Water quality in ponds for aquaculture. Alabama Agricultural Experimental Station Auburn University. Birmingham Al, USA, 482pp Duray, M.N. 1994. Daily rates of ingestion on rotifers and artemia nauplii by laboratoryreared grouper larvae of Epinephelus suillus. Philippine Scientist, 31:32-41. Duray, M.N., Estudillo, C.B., and Alpasan, L.G. 1997. Larval rearing of Epinephelus suillus under laboratory condition. Aquaculture, 150:63-76 Duray, M.N., Estudillo, C.B., and Alpasan, L.G. 1996. The effect of background color and rotifer density on rotifer intake, growth and survival of grouper Epinephelus suillus larvae. Aquaculture, 146:217-224. De Jesus, E.G.T., Toledo, J.D., and Simpas, M.S. 1998. Thyroid hormones promote early metamorphosis in grouper Epinephelus coioides larvae. General Comprehensive Endocrinology, 112:10-16.

Table1. Mean survival, body weight (wet weight in mg) and total body length (mm) of grouper fry reared in tanks with and without Kiko treatment. Experiments were done in 3-ton circular tanks with three replications. Runs Preliminary I II Treatments With Kiko With Kiko With Kiko Body weight (mg) Initial 2.78 2.78 2.47 2.47 2.18 2.18 Final 12.4±0.001 10.6±0.007 11.70±0.002 13.60±0.005 6.75±0.001 7.07±0.006 Total body length (mm) Initial 2.71±0.12 2.71±0.12 2.48±0.10 2.48±0.12 2.60±0.11 2.60±0.12 Final 7.66±0.001 7.72±0.084 9.30±0.008 9.70±0.005 7.47± 0.006 7.64±0.001 Survival (%) 0.79±0.04 0.40±0.004 0.36±0.03 0.91± 0.01 0.038±0.004 0.057± 0.001 Table 2. Mean body weight (mg), total body length (mm), weight gain, increase in total body length and survival of grouper fry reared in tanks with Kiko and control. Initial weight, 2.18mg; initial length, 2.82mm. Experiment was carried out in 5-ton tanks and duration of the experiment was 43 days (Feb. 4- March 21, 2014). Treatment Final weight (mg) Weight gain (%) Final length (mm) Increase in length (%) Survival (%) KIKO Tech 73.73 3282 17.39 516 0.115 54.53 2401 15.71 457 0.109 n=8

Figure 1. Mean body weight (mg) of E. fuscoguttatus fry reared in tanks with and without Kiko. Duration of the larval rearing Feb. 4, 2014- March 21, 2014.

Table 3. Mean temperature, salinity, ph and dissolved oxygen levels during the larval rearing runs. Runs Preliminary I II Parameters With Kiko Treatments With Kiko With Kiko Temperature AM 27.5 27.6 27.4 27.3 24.7±0.03 24.7±0.05 PM 27.7 27.7 27.9 27.8 25.1±0.02 25.2±0.04 Salinity 31.9 31.9 32.4 32.4 33.9±0.05 33.6±0.56 (ppt) ph 8.0 8.1 8.5 8.5 8.7±0.01 8.7±0.01 Dissolved Oxygen (ppm) 7.42 8.02 2.7± 2.7± 2.9±0.02 2.9±0.03 Table 4. Mean ammonia-nitrogen and nitrite-nitrogen levels during the larval rearing runs. Runs Preliminary 1 II Treatments Parameters With Kiko With Kiko With Kiko Ammonianitrogen(ppm) 0.673±0.23 1.07± 0.55 0.988±0.24 0.857± 0.32 0.581±0.12 0.636± 0.16 Nitrite- Nitrogen(ppm) 0.144±0.26 0.165± 0.29 0.071±0.08 0.061± 0.08 0.015±0.01 0.020 ±0.01 Sample size=6

Table 5. Mean ammonia-nitrogen and nitrite-nitrogen levels determined during the larval rearing of grouper (3 rd run). Culture period 45 days. Treatment Parameters With Kiko Ammonia-nitrogen 0.581 ± 0.137 0.658±0.152 Nitrite-nitrogen 0.017 ± 0.007 0.013±0.005 Sample size=8 Table 6. Water parameters determined during the larval rearing of grouper 3 rd run (Feb-March 2014. Treatments Parameters With Kiko Temperature AM 25.47 25.39 PM 26.10 27.28 Salinity (ppt) 31.71 31.73 ph 8.70 8.70 Dissolved Oxygen (ppm) 5.89 6.02