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Journal of Survey in Fisheries Sciences 2(2)17-33 2016

Reproductive performance of seahorse, barbouri (Jordan and Richardson 1908) in control condition

Nur F.A.H.1; Christianus A.1,2*; Muta Harah Z.2; Ching F.F. 3; 3 2 3 Shapawi R. ; Saad C.R. ; Senoo S.

Received: July 2015 Accepted: December 2015

Abstract Hippocampus barbouri is one of the seahorse found in shallow water of Malaysia. It is used as known as a global trade species in ornamental industry. To date, there is no documented report on seahorse aquaculture especially for H. barbouri in Malaysia even in Southeast Asia region. Seahorse aquaculture should be considered as an alternative source of seahorses to reduce the pressure on wild population. Therefore, this study was conducted to establish suitable techniques for broodstock maintenance and reproduction by focusing on culture system and feeding. Hippocampus barbouri were maintained and bred successfully in a controlled culture system. Minimum water depth required for the spawning of H. barbouri is 38 cm. Best reproductive performances was observed in broodstock fed with post-larvae shrimp. However, frozen mysid can also be used in the culture of H. barbouri. The minimal requirement of n-3 and n-6 fatty acids for the reproduction of H. barbouri was 5.13 ± 0.04 % and 14.83 ± 0.10 % respectively.

Keywords: Hippocampus barbouri, Culture system, Feeding

1- Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ] 2- Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. 3- Borneo Marine Research Institute, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia. *Corresponding author's email: [email protected]

18 Nur et al., Reproductive performance of seahorse, Hippocampus barbouri in…

Introduction in captivity is still at infancy stage with Seahorses belong to the husbandry problems and high juvenile family includes , pipehorses mortalities (Vincent, 1996; Planas et and seadragons (Lourie et al., 1999). al., 2008). In the process to develop of Harvesting seahorses to fulfill the high suitable techniques for broodstock demands for traditional Chinese maintenance and reproduction, special medicine (TCM) and marine focus was given to culture system and trade resulted in the decline of the wild feeding (Planas et al., 2008). Culture populations ( Vincent, 1996; Lourie et system is an important factor al., 1999; Foster and Vincent, 2004). particularly to maintain water quality in Seahorses also an icon for issues related order to provide favorable conditions to incidental by-catch and habitat loss for seahorse broodstock (Koldewey, (CITES, 2002). Therefore, seahorses 2005; Planas et al., 2008). are among the fourteen bony Based on previous study, diets for listed in Appendix II of the Convention seahorse broodstock have great on the International Trade of influence on gonad development and of Wild Fauna and brood size (Wong and Benzie, 2003; Flora (CITES, 2002) and Red List of Sheng et al., 2006). Adult Artemia were International Union for Conservation of regularly given to seahorse broodstock Nature as Threatened Species (IUCN, in captivity compared to other variety 2006). of food items, including mysid shrimp, Ten species of seahorses were found shrimps and amphipods either as live or in Malaysia, this includes Hippocampus frozen diet (Woods and Valentino, barbouri (Lim et al., 2011). Its shallow 2003; Dzyuba et al., 2006; Lin et al., water habitat in Malaysia exposed this 2006; Lin et al., 2007; Buen-Ursua et species to human activities (Choo and al., 2015). Providing a diet that fulfill Liew, 2004). As a global trade species, nutritional requirement is considered as H. barbouri is the most common a great challenge in seahorse species kept in aquarium due to its aquaculture. uniqueness in appearance and attractive Success reproduction is crucial to color variation from white, pale yellow ensure the sustainability of seahorse to pale brown (Kuiter, 2000; Koldewey aquaculture (Payne, 2003; Lin et al., and Martin-Smith, 2010; Olivotto et 2007). To date, no report on seahorse al., 2011). aquaculture in Malaysia and the Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ] Seahorse aquaculture can be Southeast Asian region (Koldewey and considered as an alternative source in Martin-Smith, 2010). Therefore, this order to reduce the pressure on wild study was conducted to establish population (Payne and Rippingale, breeding technology for H. barbouri 2000; Lin et al., 2007; Faleiro et al., which will contribute to the 2008). However, production of seahorse development of seahorse aquaculture.

Journal of Survey in Fisheries Sciences 2(2) 2016 19

Materials and methods barbouri. Each tank was equipped with Acquisitions of seahorse Classica® Crystal Hang-on Filter (EF- Adult seahorses were bought from 087). and sintered glass fishermen at Semporna coast of Sabah, Biohome® Plus were used as filter Malaysia. These seahorses were media. In IBS, rectangular fiberglass conditioned for 1 month before being tanks (0.4×0.4×0.5m) were used with used for breeding trials. Tilapia fry and setup similar to BMRI. Glass tanks shrimp postlarvae were used as feed (0.4L×0.3W×0.4H m) were used as during this conditioning period. Only breeding tank in IBS. Each of these healthy seahorses were used for all tanks was equipped with hang-on filter experiments. (with sponge and Biohome as filter media), similar to the setup at DoA. Culture system Figure 1, 2 and 3 shows the front view Breeding experiments were conducted of both conditioning and breeding tank at three different locations, Borneo set up in BMRI, DoA and IBS Marine Research Institute, Universiti respectively. In each conditioning and Malaysia Sabah, Kota Kinabalu, Sabah, breeding tank, plastic chain tied to a Malaysia (BMRI); Department of sinker served as holdfast for seahorses. Aquaculture, Universiti Putra Malaysia, After conditioning for 1 month, each Serdang, Selangor, Malaysia (DoA) and pair of broodstock with average Institute of Bioscience, Universiti Putra standard length (SL) 12.040±0.459cm Malaysia, Serdang, Selangor, Malaysia was transferred into breeding tank. (IBS). Different culture systems were Standard length was determined by used in each location for the measuring the length from the tip of the conditioning and breeding of wild H. tail to the mid-point of the cleithral ring barbouri. plus the length from the tip of the snout In BMRI, broodstock were to the mid-point of the cleithral ring conditioned in one tonne black circular (Lourie et al., 1999). Seahorses were polyethylene tank (H=0.8m). This blotted using filter paper before being conditioning tank was connected with weighed to get the wet weight, WW another tank of similar size as a (Job et al., 2002). Data on reproductive filtration unit filled with hard as performance was collected only after filter media. Airlift technique was used two weeks of introduction into breeding to circulate water from one tank to the tank. This experiment was carried out Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ] other. For breeding tank, similar system for 3 months. as conditioning tank was set using Prior to usage, seawater undergoes square fiberglass tank (0.4×0.4×0.4m). serial filtration (cartridge pore size of 5, In DoA, plastic tanks 1 and 0.1µm) then passed through (0.55×0.25×0.33m) were used for both Atman® UV Sterilizer (Model UV- conditioning and breeding of H. 11W) with water flow rate of 10 l/min.

20 Nur et al., Reproductive performance of seahorse, Hippocampus barbouri in…

Broodstock was fed twice daily, ad twice a week. Food consumption in the libitum with frozen Hikari® Bio-Pure percentage of body weight (% BW) was Mysis Shrimp at 0930 and post-larvae estimated by measuring the amount of of white shrimp and red tilapia fry at food intake per total weight of seahorse 1530. Faeces and excess feed were individual per day siphoned daily before and after feeding. (individual/seahorse/day). Experiment Water depth in the tank was maintained was carried out for three months. throughout the experimental period. Quantity of diets fed to seahorse was YSI Professional Plus Multi-Parameter estimated based on the data of food and HACH® DR/2400 Portable consumption. Throughout this study, Spectrophotometer were used to water quality was monitored and data measure dissolved oxygen, DO (ppm), on reproductive performance were temperature (°C), salinity (ppt), pH, recorded. ammonia (ppm), nitrate (ppm), and nitrite (ppm) twice a week throughout Diet preparation this experiment. Newly hatched Artemia nauplii from Bio Marine Artemia cysts were grown Feeding experiment in circular fiberglass tank with vigorous Experiment conducted at IBS used aeration at stocking density 200/l. similar breeding tank set up as in the Artemia nauplii were fed with rice flour previous experiment. After one month and Spirulina powder (Josens) until conditioning, each pair of broodstock reach adult stage (total length, TL: 5- with average SL and WW 10mm). Adult Artemia was enriched (12.100±0.424cm and 5.551±0.322g) with cod liver oil for twelve hours prior were introduced into each breeding to feeding. tank. Broodstock were fed with adult Both PLS and TF bought from fish Artemia (AA), frozen Hikari® Bio-Pure farms once a month were maintained in Mysis Shrimp (FM), post-larvae of one tonne rectangular fiberglass tank. white shrimp (PLS) and fry of red The average TL of PLS (the tip of tilapia (TF) twice daily at 0930 and rostrum to the tip of telson) and TF (the 1530. tip of closed mouth to the extended tip Diets were rinsed few times with of the caudal fin) that were fed to freshwater and blotted using filter paper seahorse broodstock were 13±2mm and to measure the WW of the diets prior 6±2mm respectively. Prior to use, TF Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ] fed to the seahorse broodstock. After an fry fed with newly hatched Artemia hour of feeding, excess diets were nauplii ad libitum for five minutes. siphoned, blotted using filter paper and Freshwater was used to defrost and weighted to determine daily food intake clean FM. All diet were rinsed with of seahorses broodstock. Sampling for filtered freshwater and seawater for 3 weight of broodstock was conducted times before being fed to the

Journal of Survey in Fisheries Sciences 2(2) 2016 21

broodstock. Samples of three diets were yolk sacs was estimated based on length (L) and width (W) measures, using the stored at -20°C for proximate analysis volume of an ovoid, V=1×πLWW and fatty acid profiling. 6 (Faleiro et al., 2008). Data collection

Data collected were used to calculate Proximate Analysis the weight gain (WG) and specific Proximate analysis (protein, lipid, ash growth rate (SGR) during the and moisture) of diet samples were experiment. WG(%)=(W −W )/W ×100 f i i conducted according to standard and SGR(%)=((lnW −lnW )/t)×100 f i methods AOAC. where, W is the final weight (g), W is f i the initial weight (g), SL is the standard Fatty acid profiling length (cm), and t is the duration (d). Fatty acid profiling was carried out To measure the reproductive respectively after total lipid performance of seahorse, data on the determination. Lipid from sample was occurrence of spawning, unsuccessful extracted using Soxhlet extraction spawning (when all of unfertilized apparatus. Fatty acid methyl esters found at tank bottom), pregnancy (FAME) were injected into a capillary period (number of days male incubated column SUPELCO SP-2380 (100m the fertilized eggs until giving birth), fused silica, 0.25mm internal diameters) brood size (number of newborn juvenile installed in a HP-5890 Series II Plus gas seahorse), size of newborn juvenile (SL chromatograph. Peaks were identified and WW), number of unfertilized eggs by comparison with standard FAME as (from unsuccessful spawning or some an internal standard. of unfertilized eggs aborted during spawning), number of aborted eggs Data analysis (fertilized eggs being aborted from Data were presented as mean ± standard brood pouch during pregnancy) and deviation (SD). Data collected during number of premature juvenile were the feeding experiment were analysed recorded. The newborn juvenile using one way of Analysis of Variance seahorses were cultured for two weeks. (ANOVA) and Tukey test to determine Survival was recorded to determine the the significant difference between the effect of broodstock diet on juvenile treatments. Correlation coefficient of quality. reproductive performance to selected Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ] Unfertilized eggs found at the bottom biochemical parameters were analyzed of the tanks were siphoned and counted. using correlation test at a=0.05. All Eggs diameter was measured using statistical analyses were carried out ocular micrometer fits in eyepiece of using SAS 9.4 Leica® DM500 Compound Microscope. Volume of the unfertilized eggs and

22 Nur et al., Reproductive performance of seahorse, Hippocampus barbouri in…

Results set up and data on reproductive Culture system performance between different Different culture systems used at locations. Besides the variation of tank different location depend on the set up, feed may influence the availability of materials (tank, filter, reproductive performance of seahorse. filter media). In this study, adult H. Therefore, the next experiment was barbouri were maintained and bred in conducted to determine the actual captive conditions using different effects of provided diet on the culture system. However, gas bubble reproductive of H. barbouri. disease (GBD) occurs frequently in air- lift culture system at BMRI. Feeding experiment Conditioning of H. barbouri in DoA Post-larvae of white shrimp (PLS) was with high stocking density resulted in the most preferred feed since highest level of ammonia, nitrate and broodstock of H. barbouri consumed nitrite compared to other locations. 9.81 ± 0.77% by body weight of PLS Table 1 shows the comparison in tank daily, which was significantly higher set up and water quality data between (p<0.05) compared to AA the three different locations. (5.80±0.14%), FM (5.83±0.37%) and Prior to spawning, TF (4.11±0.28). Broodstock fed with behaviour was observed mostly early PLS recorded significantly higher morning for all types of culture (p<0.05) of WG (21.30±0.67%) and systems. Males initiated the courtship SGR (2.76±0.08%) compared to by brightening their body coloration, broodstock fed with other diets (Table pushing their belly forwards to extend 3). the brood pouch, hooked the tail of Broodstock fed on PLS and FM have female and swim in pair. Upon the highest number (p<0.05) of successful spawning, female will spawning occurrences (Table 4). transfer eggs into brood pouch of male. However, with regards to brood size, Transfers of eggs were observed to be broodstock fed on PLS produced largest unsuccessful in GBD male or when the size (p<0.05) juveniles (SL: 0.95±0.05; water depth is less than 0.3m. WW: 0.005±0.001) and highest number Breeding tank set up in IBS with of juvenile (384±76.37 pieces). water depth 0.38m recorded the least Broodstock fed with AA recorded occurrences of unsuccessful eggs significantly lowest (p<0.05) brood size Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ] transfer. As for water quality, lowest producing significantly higher (p<0.05) ammonia (0.04±0.03 ppm), nitrate number of premature eggs (21.5±3.54 (1.15±0.40 ppm) and nitrite (0.02±0.02 eggs). These eggs were fertilized eggs ppm) level were recorded for the that have been aborted before breeding tank set up in IBS. Table 2 completely develop into juvenile. shows the comparison on breeding tank Highest number of abnormal juvenile

Journal of Survey in Fisheries Sciences 2(2) 2016 23

was produced by broodstock fed on AA PLS produced significantly highest (24.5±7.78 pieces) or TF (27.5±10.61 (p<0.05) volume of eggs and yolk pieces). Abnormal juvenile was which were 5.36±0.72 µL and characterized by short snout and 4.67±0.67µL respectively compared to incompletely developed of the other treatment. rays which lead to swimming inability. Proximate compositions for each Male of H. barbouri in all treatment type of diet were presented in Table 5. incubated the fertilized eggs for 14 days Percentage of protein was highest before give birth the newborn juvenile. (66.51±0.48%) in PLS and TF Unsuccessful spawning occurs to the (65.60±1.98%). Significantly highest broodstock fed with AA when female fat (17.19±0.31%) and energy H. barbouri failed to transfer eggs into (227.00±0.00kJ) were recorded in TF. male brood pouch. No unsuccessful Carbohydrate (29.03±2.31%) and ash spawning occurs in the broodstock fed (25.38±1.02%) content was with other diets. Significantly higher significantly highest in AA. numbers of unfertilized eggs (55±4.24 Fatty acid profiles of different diet eggs) was found at the tank bottom were recorded in Table 6. Palmitic acid consist of broodstock fed with AA (C16:0) was significantly highest compared to the broodstock fed on TF (p<0.05) in AA. Mono unsaturated fatty (5±0 eggs), FM (4±0 eggs) and PLS acid (MUFA) especially oleic acid (3±0 eggs) respectively. Shape of (C18:1-n9) was significantly higher unfertilized eggs varies from rod, (p<0.05) in FM. Significantly highest teardrop to round. Unfertilized eggs percentage of total n-3 (16.70±0.04%) consist of yolk surrounded by orange- and n-6 (24.92±0.20%) fatty acid was yellow oil droplets. Broodstock fed on observed in TF and PLS respectively.

Table 1: Data during conditioning broodstock of Hippocampus barbouri in three different locations. BMRI DoA IBS Water depth 0.75 m 0.30 m 0.48 m Tank Size (m) π × 0.75 × 0.80 0.55 × 0.25× 0.33 0.4× 0.4 × 0.5 πr2H (L × W × H) (L × W × H) Colour Black Transparent Blue Material Polyethylene tank Plastic tank Fiberglas tank Filtration System Air-lift Hang-on filter Air-lift Media Hard coral Sponge & Bio-home Hard coral Stocking density 1 ind/ 80l 1 ind/ 10l 1 ind/ 18l

Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ] Feeding frozen Hikari® Bio-Pure Mysis Shrimp, red tilapia fry & post-larvae of white shrimp Water parameter DO (ppm) 7.46 ± 0.20 4.94 ± 0.34 7.25 ± 0.11 Temperature (°C) 27.65 ± 0.64 26.78 ± 0.53 28.03 ±1.44 Salinity (ppt) 31.84 ± 0.38 30.58 ± 0.52 30.89 ±0.53 pH 8.93 ± 0.20 7.95 ± 0.21 7.98 ±0.18 Ammonia (ppm) 0.10 ± 0.06 0.20 ± 0.09 0.04 ±0.03 Nitrate (ppm) 2.07 ± 0.35 4.03 ± 2.03 1.15 ±0.40 Nitrite (ppm) 0.06 ± 0.03 0.16 ± 0.07 0.02 ±0.02

24 Nur et al., Reproductive performance of seahorse, Hippocampus barbouri in… Table 2: Data on breeding of Hippocampus barbouri at three different locations. BMRI DoA IBS Water depth 0.35 m 0.30 m 0.38 m Tank Size (m) 0.4L × 0.4W × 0.4H 0.55L × 0.25W × 0.33H 0.4L × 0.3W × 0.4H Colour Blue Transparent Transparent Material Fiberglass tank Plastic tank Glass tank Filtration System Air-lift Hang-on Hang-on Media Hard coral Sponge & Bio-home Sponge & Bio-home Stocking density 1 ind/ 28l 1 ind/ 20l 1 ind/ 22l Feeding frozen Hikari® Bio-Pure Mysis Shrimp, red tilapia fry & post-larvae of white shrimp Water DO (ppm) 7.41 ± 0.16 5.25 ± 0.23 5.80 ± 0.07 parameter Temperature (°C) 27.88 ± 0.85 27.03 ± 0.24 29.17 ± 0.98 Salinity (ppt) 31.88 ± 0.38 29.83 ± 0.92 30.47 ± 0.80 pH 7.98 ± 0.15 7.76 ± 0.17 8.16 ± 0.10 Ammonia (ppm) 0.10 ± 0.04 0.13 ± 0.02 0.04 ± 0.01 Nitrate (ppm) 1.23 ± 0.43 2.38 ± 0.42 1.58 ± 0.52 Nitrite (ppm) 0.03 ± 0.02 0.05 ± 0.02 0.02 ± 0.01 Reproductive Spawning occurrence 13 10 20 performance Unsuccessful 2 2 - spawning Incubation period 14-15 days 14-15 days 13-14 days Brood size 54.77 ± 5.183 26.70 ± 9.129 59.05 ± 4.947 Unfertilized eggs 93 114 2 Juvenile size SL:0.83 ± 0.02 cm SL:0.81 ± 0.03 cm SL: 0.85 ± 0.02 WW: 0.004 ± 0.001 g WW: 0.004 ± 0.001 g WW: 0.005 ± 0.001 g No. of premature - 23 -

Table 3: Data on food consumption, weight gain (WG) and specific growth rate (SGR) of Hippocampus barbouri fed on different feed. Treatment Amount of feed consumed WG (%) SGR (%) Wet weight (g) Percentage by body weight (%) Adult Artemia 0.30 ± 0.01b 5.80 ± 0.14b 9.28 ± 0.28b 1.27 ± 0.04b Frozen mysids 0.32 ± 0.01b 5.83 ± 0.37b 12.29 ± 1.74b 1.65 ± 0.22b Post larvae shrimp 0.55 ± 0.02a 9.81 ± 0.77a 21.30 ± 0.67a 2.76 ± 0.08a Tilapia fry 0.23 ± 0.02c 4.11 ± 0.28c 10.88 ± 0.82c 1.48 ± 0.11c Different superscript letters shows significant differences between treatment at p<0.05.

Table 4: Data on reproductive performance of Hippocampus barbouri fed with different feed. Adult Artemia Frozen Mysids Post Larvae Shrimp Tilapia Fry Spawning occurrence 2 ± 0c 5 ± 0a 5.5 ± 0.71a 3.5 ± 0.71b Unsuccessful spawning 1 ± 0 - - - Brood size 74 ± 11.31c 211 ± 2.83b 384 ± 76.37a 143 ± 26.87bc Unfertilized eggs 55 ± 4.24a 4 ± 0b 3 ± 0b 5 ± 0b No. of premature 21.5 ± 3.54a 11 ± 0b - 7 ± 0b No. of abnormal 24.5 ± 7.78a 7.5 ± 0.71b - 27.5 ± 10.61a volume (µL) 1.60 ± 0.30c 2.52 ± 0.41b 5.36 ± 0.72a 2.02 ± 0.31c c b a b

Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ] Yolk volume (µL) 0.98 ± 0.09 1.75 ± 0.42 4.67 ± 0.67 1.39 ± 0.14 Juvenile size SL: 0.77 ± 0.03b SL: 0.73 ± 0.03b SL: 0.79 ± 0.04b SL: 0.95 ± 0.05a (SL, cm; WW, g) WW: 0.004 ± WW: 0.003 ± 0.000c WW: 0.004 ± 0.000b WW: 0.005 ± 0.001a 0.000b Juvenile survival (%) 76.67 ± 7.64c 91.67 ± 2.89b 99.00 ± 1.00a 81.67 ± 2.89c Different superscript letters shows significant differences between treatment at p<0.05. SL = standard length; WW = wet weight.

Journal of Survey in Fisheries Sciences 2(2) 2016 25

Table 5: Data on proximate composition of different feed. Treatment Percentage by dry matter (%) Moisture (%) Energy (kJ) Protein Fat Carbohydrate Ash Adult Artemia 92.73 ± 0.04a 33.28 ± 3.31c 12.31 ± 0.03b 29.03 ± 2.31a 25.38 ± 1.02a 110.91 ± 2.96d Frozen mysids 91.42 ± 0.00b 60.14 ± 2.15b 4.72 ± 0.09c 21.56 ± 1.49b 13.58 ± 0.58b 134.00 ± 0.00c Post larvae 87.24 ± 0.05d 66.51 ± 0.48a 3.68 ± 0.13d 11.67 ± 0.62c 18.14 ± 0.01c 185.00 ± 0.00b Shrimp Tilapia fry 87.84 ± 0.14c 65.60 ± 1.99ab 17.19 ± 0.31a 7.10 ± 2.07d 10.12 ± 0.23d 227.00 ± 0.00a

Different superscript letters shows significant differences between treatment at p<0.05.

Table 6: Data on fatty acid profile of different feed. Fatty acid Treatment Adult Artemia Frozen mysids Post larvae shrimp Tilapia fry Myristic acid, C14:0 7.97 ± 0.01a 3.64 ± 0.01b 1.59 ± 0.03d 2.32 ± 0.04c Palmitic acid, C16:0 40.66 ± 0.01a 30.14 ± 0.00b 18.21 ± 0.16c 30.25 ± 0.08b Palmitoleic acid, C16:1n-7 5.03 ± 0.01b 3.61 ± 0.08d 5.76 ± 0.07a 4.44 ± 0.04c Stearic acid, C18:0 14.71 ± 0.01c 18.22 ± 0.19a 16.16 ±0.08b 12.43 ± 0.07d Oleic acid, C18:1n-9 21.57 ±0.03c 24.46 ± 0.04a 19.94 ± 0.02d 23.51 ± 0.03b Linoleic acid, C18:2n-6 4.90 ± 0.04d 14.61 ± 0.08b 22.63 ± 0.23a 8.04 ± 0.04c Linolenic acid, C18:3n-3 1.49 ± 0.03c 1.88 ± 0.05b 2.71 ± 0.02a 0.58 ± 0.01d Arachidonic acid, C20:4n-6 (ARA) 0.00 ± 0.00c 0.23 ± 0.02b 2.29 ± 0.04a 2.32 ± 0.04a Eicosapentaenoic acid, C20:5n-3 1.80 ± 0.06b 1.27 ± 0.04c 5.99 ± 0.04a 5.73 ± 0.05a (EPA) Docosahexaenoic acid, C22:6n-3 1.89 ± 0.01c 1.99 ± 0.05c 4.72 ± 0.03b 10.40 ± 0.01a (DHA) n-3 5.17 ± 0.08c 5.13 ± 0.04c 13.42 ± 0.06b 16.70 ± 0.04a n-6 4.90 ± 0.04d 14.83 ± 0.10b 24.92 ± 0.20a 10.35 ± 0.00c n-3: n-6 1.06 ± 0.02b 0.35 ± 0.01d 0.54 ± 0.01c 1.62 ± 0.01a DHA: EPA 1.05 ± 0.03c 1.57 ± 0.02b 0.79 ± 0.00d 1.82 ± 0.02a DHA: ARA 0.00 ± 0.00d 8.77 ± 0.88a 2.07 ± 0.02c 4.50 ± 0.08b Different superscript letters shows significant differences between treatment at p<0.05. n-3 =omega 3; n-6 =omega 6. Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ]

26 Nur et al., Reproductive performance of seahorse, Hippocampus barbouri in…

Figure 1: Front view of A, conditioning tank and B, breeding tank in BMRI. PVC pipe (Ø = 5cm) and air-lift technique were used to circulate water from one tank to another.

Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ]

Figure 2: Front view of both conditioning tank and breeding tank in DoA. Hang- on with sponge and sintered glass Biohome® Plus were used to filtered water.

Journal of Survey in Fisheries Sciences 2(2) 2016 27

Figure 3: Front view of A; conditioning tank and B; breeding tank in IBS.

Discussion in air-lift system creates water Development of suitable techniques movement as well as supplemental especially the physical factor is very aeration to minimize the consumption important for broodstock maintenance of resources (Loyless and Malone, and reproduction (Planas et al., 2008). 1998). However, it causes the presence Culture system is the physical key of air bubble in culture system and factor to provide favourable conditions increases the occurrence GBD in with minimal resources ( Duarte et al., seahorses (Reinemann, 1987; Planas et 2011; Blanco et al., 2014). Based on al., 2008). this study, H. barbouri can be Super saturation of gas especially maintained in different culture system. nitrogen and oxygen typically related Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ] However, there are some factors with the application of air-lift technique constraining the successful spawning of (Parker et al., 1984). This super H. barbouri in captive conditions. saturation become the causative agent Unsuccessful spawning in the present for GBD in seahorse, whereby gas study frequently relates to the entrapment in the brood pouch, occurrence of GBD in seahorse cultured subcutaneous emphysema on the tail

28 Nur et al., Reproductive performance of seahorse, Hippocampus barbouri in…

segment or hyperinflation of swim broodstock in this study due to its ready bladder (Koldewey, 2005; Planas et al., supply. Furthermore, Garcia et al. 2008; Koldewey and Martin-Smith, (2012) reported the use of fish larvae as 2010). During this study, air-lift system feed for adult H. barbouri. recorded the highest dissolved oxygen, Broodstock of H. barbouri fed on to near saturation level as suggested by PLS shows the best reproductive Masser et al. (1999). performance with high numbers of Tank height is another restrictive spawning occurrences and brood size. factor to the successful breeding of H. Coincidentally, it was the most barbouri. It is important to ensure water preferred feed by the seahorses as depth is sufficient to enable successful compared to the other feeds. Based on eggs transfer to the male brood pouch optimum foraging theory, seahorse during spawning process (Woods, prefer to consumed caridean shrimp 2003; Koldewey, 2005). Hippocampus with lowest energy expenditure abdominalis requires tank depth of at required (Anderson Jr, 2000; Felício et least 90 cm to facilitate the egg transfer al., 2006). In addition, structure of (Sobolewski, 1997). However, high syngnathid eyes made them adaptable water level will cause difficulty in tank to the mobility and carotenoid-rich prey maintenance. Therefore, it is important (Collin and Collin, 1999). Similar to to determine minimum water depth previous finding, adult H. barbouri required to ensure successful eggs prefers to consumed FM compared to transfer during seahorse spawning. the highly mobile AA and TF (Felício Breeding tank with different water et al., 2006). depth was tested in this study. All Quality and quantity of feed give a seahorse broodstocks spawned major influence on brood size, which successfully in tank with 38 cm depth affects their gonad development and in IBS. Hence, H. barbouri required at sperm quality (Wong and Benzie, 2003; least 38 cm water depth for successful Foster and Vincent, 2004; Lin et al., eggs transfer. 2007). According to Otero‐Ferrer et al. Adequate of nutrients is one of the (2012) the utilization of live feed main factors influencing the spawning resulted in better growth and gonad outcome of fish (Izquierdo et al., development of adult seahorses. 2001). Variety of feeds were given to However, in this study broodstock fed seahorse broodstock in captivity which with FM showed better reproductive Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ] include adult Artemia, mysid shrimp, performance (in terms of spawning amphipods and shrimps, given as live occurrence, brood size and juvenile or frozen (Woods and Valentino, 2003; survival) as compared to broodstock fed Dzyuba et al., 2006; Lin et al., 2007; with live feed such as AA and TF. This Palma et al., 2012). In this study, TF condition may due to the higher was selected as feed for seahorse

Journal of Survey in Fisheries Sciences 2(2) 2016 29

percentage of FM consumed by H. amphidromous marine fish contained barbouri as compared to AA and TF. higher n-6 compared to n-3 (Usman, Composition of n-3 HUFA, 2014). In East Malaysia, H. barbouri especially DHA considered as the usually found together with H. kuda at major dietary requirement for the or near the river mouths successful reproduction in marine fishes (Choo and Liew, 2004). Therefore, H. by affecting steroidogenesis, barbouri may be an amphidromous spermiation and other reproductive marine fish that requires more n-6 parameters (Izquierdo et al., 2001). compared to n-3. Highest percentage of DHA plays an important role in cells protein and n-6 found in PLS compared membrane by regulating the integrity to other feed offered in this study may and function besides being an important likely be the reason of better component of phosphoglycerides in reproductive performance of H. gonad and juvenile (Izquierdo et al., barbouri. 2001). Its deficiencies can cause H. barbouri can be maintained and decreases in fecundity, lower bred in captive condition in Malaysia. fertilization rates, embryo deformities However, presence of bubble gas in and poor larval quality (Izquierdo et al., culture system should be avoided since it can be a causative agent for GBD in 2001; Otero‐Ferrer et al., 2012). In contrast, the present study, broodstock seahorse. Minimum water depth H. barbouri fed on PLS (which lower in required for successful spawning of H. n-3 contents) compared to TF, shows barbouri is 38 cm. Based on this study, better reproductive performance evident the best diet for H. barbouri broodstock with bigger brood size with no is PLS, obvious with the best abnormality on newborn juveniles. Low reproductive performance. However, n-3 content in live feed may be FM can also be used in the culture of H. sufficient to compensate the dietary barbouri. The minimal requirements of n-3 and n-6 fatty acids for reproduction requirement for seahorse (Otero‐Ferrer et al., 2012). In addition, low n-3 of H. barbouri are 5.13±0.04% and content also found in eggs of H. 14.83±0.10% respectively. Further guttulatus (Planas et al., 2008) study on bioavailability and n-6 Composition of protein in feed also requirements for H. barbouri should be affects the reproductive performance of conducted. marine fish (Izquierdo et al., 2001; Downloaded from sifisheriessciences.com at 15:21 +0330 on Tuesday September 28th 2021 [ DOI: 10.18331/SFS2016.2.2.2 ] Buen-Ursua et al., 2015). For example, Acknowledgement the reduction in protein composition Author would like to thank the Ministry reduced egg viability in seabass (Cerdá of Higher Education Malaysia for et al., 1994). Often freshwater fish funded this study through Fundamental contained higher n-6 compared to n-3 Research Grant Scheme (FRGS). (Steffens, 1997). However, some

30 Nur et al., Reproductive performance of seahorse, Hippocampus barbouri in…

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