Bone formation and growth of climbing perch Anabas testudinieus larvae fed with Zn enriched Artemia nauplii

The aim of this study was to determine the optimum level of Zinc (Zn) enrichment in Artemia sp. nauplii as a live feed to improve bone formation and growth of climbing perch Anabas testudinieus larvae. The study consisted of four different Zn enrichment levels (0.0, 0.05, 0.1, and 0.15 mg/mL) in Artemia sp. nauplii. The enrichment was performed for 12 hours with the nauplii density of 1 ind/mL. Climbing perch larvae with an average initial length of 1.65 ± 0.15 mm were fed four times daily with the enriched nauplii. For the first 5 days, all larvae were fed with rotifer followed by feeding with enriched Artemia nauplii ad libitum. The results showed that the application of Artemia sp. enrichment at 0.1 mg Zn/mL influenced the bone formation, increased the growth, and improved the fish survival of climbing perch larvae. Feeding with 0.1 mg/mL Zn enriched Artemia nauplii could be recommended as a strategy to improve the bone formation and growth performance of climbing perch larvae.


INTRODUCTION
Climbing perch Anabas testudinieus is a freshwater fish that is potentially cultured due to a high economical value, but having a slow growth (Kohinoor et al., 2012;Morioka et al., 2009). One factor influencing the slow growth is feed. Growth is initially begun with a bone formation. Bone formation happens in two ways, namely through the intra-membrane and endrochondral osification (Boglione et al., 1982). Bone formation needs macro and micro mineral elements. One of important micro minerals in bone formation is zinc (Zn). Zn is a mineral element that is mostly accumulated in bones compared to liver as the main organ of micro mineral preservation (Setiawati et al., 2007) and as a cofactor in bone formation (Yamaguchi, 2010). Kaliky et al. (2019) reported that Zn also played an important role in the reproduction process of striped catfish.
Commonly, Zn deficiency in feed can lead to low growth and bone demineralization, especially either on juvenile or adult fish (Baeverfjord et al., 2019). On red sea bream larvae, Zn is also functioned to improve growth and backbone formation (Nguyen et al., 2008). In osteology, Zn has anabolic effect on the osteogenesis in osteoblast (Seo et al., 2010). Excessive Zn in feed can decrease the Ca and P content in bones and body of giant gouramy fish and increase the Mn content in bones (Setiawati et al., 2007). The addition of Zn in feed should follow the requirement, thus can balance the other mineral element performances, such as Ca, P, and Mg as also important in bone formation and growth (Holt, 2011). Therefore, sufficient Zn is required for bone formation.
Fish larvae commonly requires Zn content around 20-30 mg/kg (NRC, 1993). Giant gouramy requires Zn supplementation of 25 -50 mg/kg feed (Setiawati et al., 2007). Fish larvae are commonly and initially fed with live feeds in the form of rotifers and Artemia sp. nauplii. The climbing perch larvae given rotifers on 2 days after hatching (DAH) (3.027 mm) until 10 DAH (5.4 mm), while Artemia sp. nauplii are given on 7 DAH (4.2 mm) until 20 DAH (10.23 mm) (Yulintine, 2012). The Artemia sp. nauplii are small-sized live feed with high nutrient contents (Chahyaningrum et al., 2015), Artemia sp. nauplii and rotifer have a character that can be used as initial feed for larvae based on mouth-opening and slow movement (Yulintine, 2012).

Container and materials
The container used was aquarium sized 15×15×20 cm 3 as much as 8 pieces for Zn enrichment on Artemia sp. nauplii and 20×20×30 cm 3 as much as 12 pieces for larval rearing. The climbing perch larvae used were 1.65 ± 0.15 mm and stocked around 40 ekor/L, therefore resulting 1700 larvae in total. Artemia sp. nauplii used had 12.18% proteins, 72.24% moisture, 3.22% ash, 0.70% lipids, 2.02% crude fiber, and 9.64% NFE. The Zn used was in the form of ZnSO4.7H2O.

Artemia sp. cyst hatching
Artemia sp. cysts were hatched gradually to fulfill the climbing perch larvae live feed availability. Artemia sp. cysts were hatched from 4 days after climbing perch egg hatching. Artemia sp. nauplii were hatched using water filled container with 30 g/L salinity. Then, the Artemia sp. cysts were distributed into the hatching container as much as 1 g and given an aeration for 24 hours. Artemia sp. nauplii were harvested and rinsed with freshwater, then moved into the enrichment container (Setiawati et al.,2013) and taken as much as 2 g samples of Artemia sp. nauplii for Zn mineral analysis and 10 g for proximate analysis.

Zn enrichment on Artemia sp. nauplii
The Zn enrichment on Artemia sp. nauplii was performed by following the nutrient enrichment method for live feed (Rahardja et al., 2009). The enrichment container was filled with 200 mL freshwater and 6 g salt. The hatched Artemia sp. nauplii were stocked into the available container with 1 nauplius/mL density (Setiawati et al., 2013).
Then, different dozes of Zn (0.0, 0.05, 0.1, and 0.15 mg/L) were added and dissolved with water into the enrichment container. The Artemia sp. nauplii were harvested after 12 hours (Matsumoto et al., 2009) and rinsed with freshwater, then directly given to the climbing perch larvae. The enriched Artemia sp. nauplii were taken 2 g from each treatment for Zn mineral analysis and 10 g from each treatment for proximate analysis.

Climbing perch larval rearing
The newly hatched climbing perch larvae were reared on the prepared aquaria with the stocking density of each aquarium was 120 larvae (40 larvae/L) for 18 days. before stocked, larvae were acclimatized for 15 minutes in the aquarium. Larvae were initially fed on 2 days after hatching (when the egg yolk was fully absorbed) until 5 days after hatching by giving 5 ind/ml rotifers with 4 times daily. Climbing perch larvae were given Zn-enriched Artemia sp. nauplii ad libitum with 4 times daily. The water quality during the larval rearing period was controlled at 6.5-7.0 mg/L dissolved oxygen, pH 6.9-7.0, and 28-30 o C temperature. Water quality maintenance was performed by syphonization every morning before feeding and 50% water exchange. After the climbing perch larvae were harvested, larvae were taken 20 larvae from each replication as absolute length growth samples and 3 larvae from each treatment for bone staining using Alizarin red and Alcian blue method.

Parameters
Parameters obtained were; Artemia sp. nauplii proximate analysis, Zn mineral analysis on Artemia sp. nauplii and rotifer, Zn, P, Ca, and Mg mineral analysis on fish larvae, survival rate, absolute length growth, and backbone formation.

Artemia sp. nauplii proximate
The proximate analysis result showed that Zn enriched Artemia sp. nauplii had a different proximate composition (Table 1). Table 2 showed the mineral analysis result of Zn on rotifers and Artemia sp. nauplii, besides mineral analysis of Zn, P, Ca, and Mg in fish larvae given nauplii enriched with different Zn. The Zn content on Artemia sp. nauplii enriched with Zn was higher   than without Zn enrichment. Hasil analisis mineral larva ikan menunjukkan bahwa semakin tinggi konsentrasi pengayaan Znmaka semakin tinggi kandungan mineral (Zn, P, Ca, dan Mg) pada larva ikan betok. The mineral analysis on fish larvae indicated that the higher Zn enrichment concentration given, the higher mineral contents (Zn, P, Ca, and Mg) obtained.

Backbone formation on climbing perch larvae
Artemia sp. nauplii enriched with 0.05, 0.1, and 0.15 mg/mL Zn influenced the climbing perch backbone formation (Figure 1). Based on the observation of climbing perch larvae backbone formation, the enriched Artemia sp. nauplii given with 0.1 mg/mL Zn obtained a red colored hard bone phase condition. Meanwhile, Artemia sp. nauplii without Zn enrichment only obtained a transition phase between cartilaginous to hard bone shown from the transition of transparent blue to red colored bones.

Absolute length growth and survival rate
The climbing perch larva fed Artemia sp. nauplii enriched with 0.1 mg/mL Zn had a higher absolute length growth value than control (P<0.05) ( Table 3). The highest survival rate of climbing perch larvae was obtained from larvae fed Artemia sp. nauplii enriched with 0.1 and 0.15 mg/mL Zn compared to other treatments.

Discussion
The Zn content on Artemia sp. nauplii enriched with Zn increased along with the increased concentration of Zn in the enrichment solution. This followed the previous results of Nguyen et al. (2008) and Matsumoto et al. (2009). The increased Zn content was caused as Artemia sp. nauplii could absorb available Zn better in the enrichment container. This study result showed that Artemia nauplii enriched with Zn increased the Zn concentration of larval body. This condition followed the result of Eid and Ghonim (1994), who stated that the increased Zn content in Oreochromis niloticus fish feed could also increase the Zn content in bones and growth. The increased mineral contents of P, Ca, and Mg was due to the synergetic relationship with Zn mineral. According to Setiawati et al. (2007), excessive Zn content in feed could decline P and Ca content in bones as the analysis result on giant gourami indicated that Zn in feed tended to increase the body Zn content, therefore accumulated in the body as one of which was in the bone tissues. The increased Zn mineral in climbing perch larval body influenced the backbone formation, besides survival rate and growth of climbing perch larvae. The same result was reported on gilthead seabream that showed increased growth, bone mineralization, and decreased abnormality level, when given Se, Mn, and Zn supplemented feed (Izquierdo et al., 2016).
The result indicated that Artemia sp. nauplii enriched with 0.1 mg/mL Zn could accelerate the backbone formation marked by the increased length growth of climbing perch larvae. Zn is known to induce the cell differentiation, cell proliferation, and mineralization in osteoblast (bone tissue), as well as improving the insulin-like growth factor I (IGF-I) and transforming growth factor β1 (TGFβ1) production in osteoblastic cells (Yamaguchi, 2010). The larval backbone formation is closely related to feed utilization effectivity and nutrient content completeness in feed consumed, therefore will be expressed on the morphological appearance (Aslianti et al., 2014). Bone formation that is still in the cartilaginous phase affects on the low survival rate and slow growth. Larvae on the cartilaginous phase are at high risk to be dead during the rearing period.
This study result showed that climbing perch larval growth was commonly better than the previous study. This condition happened as only taking 18 days to reach more than 12 mm length, which was faster than the study of Yulintine (2012) that required more than 20 days. The Zn enriched Artemia nauplii given produced better growth and survival rate than control. This condition indicated that Zn had a very important role in fish larval growth. A study on carp showed that the supplementation of Zn in feed could improve protein concentration in blood and intestine (Chupani et al., 2017;2018), while on giant freshwater prawn, the supplementation of Zn was reported to improve growth, survival rate, and immunity (Muralisankar et al., 2014;. Fish feed with 20 mg/kg Zn produced the highest weight and SGR and was significantly different among other treatments (P<0.05) (Mondal et al., 2020).
The survival rate value of climbing perch larvae on Artemia sp. nauplii enriched with 0.1 mg/mL Zn treatment was 64.72%, higher than the survival rate of climbing perch reported by Morioka et al. (2009) with 20% andBugar et al. (2013) with 18-31%. Artemia sp. nauplii enrichment with Zn was suspected to induce the climbing perch larval resistance. Zn also had an important role on the growth of rohu fish Labeo rohita. SGR of rohu fish was reported to improve along the increased doze of Zn until 42 mg/kg (Akram et al., 2019). The increased survival rate of climbing perch larvae was due to the normal condition occurred to maintain the movement space and utilize the feed maximally for sustaining the survival rate (Bugar et al., 2013). Moreover, the increased survival rate of climbing perch larvae was also due to the sufficient live feed given as based on the larval mouth-opening and number of feed given that could create a normal condition for larval movement space during the rearing period (Suriansyah, 2012).

CONCLUSION
The Artemia sp. nauplii given with 0.1 mg/mL Zn enrichment could increase the larval backbone formation and growth rate, as well as survival rate of climbing perch larvae.