Twelve aquariums (46 × 36 × 25.6 cm ³) was used in this research with 25 L of water for 10 fish. There were four groups of different pellets and triplicates with the extracts added and a control. Each group used 10 goldfish, the fish were obtained from Rama Jaya Mahakam Company’s hatchery in Kutai Kartanegara regency, East Borneo, Indonesia, totalling 120 fish in this experiment.

The fish sample was collected using a fish sorting bucket of size 8 cm, fish that escaped from the 8 cm fish sorting bucket were collected. The fish species was goldfish ( Cyprinus carpio), the fish sex was mixed between male and female, the developmental stage was larva size 8–9 cm, and the initial weight range was 5 ± 0.6 g.

Before the experiment, the goldfish were adapted to the natural environment for seven days and the fish were provided ad libitum access to commercial feed twice a day (at 8.00 a.m. and 4.00 p.m.). Moreover, the fish were first tested for infection by Aeromonas and Pseudomonas by incubating their isolated liver and kidneys with GSP (Himedia ^®) media; if the bacteria did not grow the fish were considered safe for use in the experiment, whereas if bacterial growth was constantly detected, the fish were soaked in 30% formalin for five minutes and the treatment was repeated for seven days until they were free from the bacteria, Aeromonas and Pseudomonas.

The method of Hardi et al. ³⁰ was used for B. pandurata extraction; the rhizome was cleaned to remove soil and then minced into pieces (0.3–0.5 cm) by using a chopper, and the chopped fingerroot was dried at 40–45 °C for 48 h in an oven. The dried fingerroot was continuously blended and soaked in 96% ethanol for 48–72 h at a 1:10 ratio (i.e. 1 kg of fingerroot powder was soaked in 10 L of ethanol), and the process was continued to extraction for 24 h until the BPE was obtained with a viscosity of 10–11.

BPE was used in goldfish feed as per the method of Hoseinifar et al., ³¹ with the following four feed pellets being applied as treatments:

Pellet 1 (control diets with BPE 0 g kg ⁻¹ fish feed).

Pellet 2 (supplemented-control diets with 2 g kg ⁻¹ fish feed of B. pandurata).

Pellet 3 (supplemented-control diets with 4 g kg ⁻¹ fish feed of B. pandurata).

Pellet 4 (supplemented-control diets with 6 g kg ⁻¹ fish feed of B. pandurata).

Goldfish feed was formulated as shown in Table 1.

The bacteria pathogen for challenges were A. hydrophila (EA-01) and P. fluorescens (EP-02) combination bacteria with bacterial density of 10 ⁵ CFU mL ⁻¹ each bacteria and injection of as much as 0.1 mL were given to each fish. The bacteria were cultured in TSB (Merck ^®) medium for 24 h in 28–30 °C. Suspense bacteria was collected and centrifuged for 15 minutes in 7000 rpm and bacteria pellet was washing with sterile water twice, and then the bacteria suspense was counting density using TPC to measure 10 ⁵ CFU mL ⁻¹, and bacteria had been properly prepared following the methods of Hardi et al. (2016).

Challenge tests were carried out at week eight after feeding with different formulations, and mortality observations were checked from 24 hours after the first injection until week 12. The rate of resistance against both bacteria was measured using RPS (Amend, 1981). Eventually, the rate of protection against pathogen bacteria was also measured at week 12.

Three parameters of water quality—temperature, pH, and dissolved oxygen (DO)—were measured (twice daily, in the morning and evening) using a multi-parameter checker, whereas total ammonia nitrogen was measured using a spectrophotometer.

Immunological parameters were evaluated by quantifying total leukocyte (TL) using cells mm ⁻³ numbers and by measuring lysozyme activity (LA) according to the method of Parry et al. ³² (with the results expressed using the unit μg mL ⁻¹). Subsequently, phagocytosis activity (“index phagocytic,” IP) the results expressed using percentage and respiratory burst activity (RBA) were examined as per the method of Van Doan et al., ³³ with a few modifications.

Goldfish growth was measured according to the method of Hoseinifar et al. ³⁴ at Weeks 4 and 8 after the feeding period; growth was measured in terms of the following criteria: weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR). These data were collected at 8 weeks after the feeding period:

WG = final weight (g) – initial weight (g);

SGR (%) = 100 × (ln final weight – ln initial weight)/duration of experiment;

FCR = feed offered (dry weight)/weight gain (wet weight).

The challenge test was administered by using A. hydrophila (EA-01) and P. fluorescens (EP-02); the bacteria were appropriately prepared as per the method of Hardi et al. ^{6 , 35} The test was administered at Week 8, with 10 goldfish being exposed to a specific treatment; the fish were infected with the combined bacteria by means of intermuscular injection (0.1 mL each fish) of 10 ⁵ CFU mL ⁻¹ of the bacteria at a 1:1 ratio. Subsequently, fish mortality was monitored from 24 h after the first injection until week 12. The rate of resistance toward both bacteria was measured by using the relative percentage of survival (RPS) value, as defined in the Amend (1981) method. Lastly, the rate of protection against infection with the pathogenic bacteria was measured at Week 12.

SR (%) = (final fish number/initial fish number) × 100;

RPS = 1 − (test mortality/control mortality) × 100.

The obtained data were analyzed for statistical significance by using MINITAB ^® 17 computer program (Minitab, RRID:SCR_014483), followed by the DUNCAN test. The average scores calculated were considered significantly different at P < 0.05.

Goldfish growth performance was measured at Weeks 4 and 8 after the feeding period. At Week 4, WG and SGR were significantly higher ( P < 0.05) after all BPE treatments than after the control treatment (no BPE) ( Table 2). The highest SGR and WG were recorded in the case of the goldfish that received fish feed containing BPE at 4 g kg ⁻¹, and these values at Week 8 were considerably different from those measured for goldfish exposed to the control and others treatments.

At Week 4, goldfish exposed to the control treatment grew by SGR 3.99–5.70 g; by contrast, treatment with BPE drastically enhanced growth, by 7.19–10.54 g in the case of the feed containing 4 g kg ⁻¹ BPE, and this was 2- or even 3-fold higher than that with the control treatment ( Table 2). Moreover, consistent results were obtained at Week 8 after the third fish feeding, with the growth doubling relative to the initial weight and being markedly distinct from that measured after the control treatment. Furthermore, besides growth, feed efficiency also increased, as demonstrated by the FCR increase (relative to control) being substantially lower (0.55) at Week 8 in the case of goldfish that were fed Pellet 3 (BPE at 4 g kg ⁻¹), and this FCR value was also significantly different from those calculated after treatment with BPE at the two other concentrations (2 and 6 g kg ⁻¹), which produced roughly equal effects.

Next, immunological parameters were measured at Weeks 4 and 8 after the feeding period ( Table 3). Activity of Lysozymes (LA) in addition to control of feed-fish in BPE was significantly higher ( P < 0.05) ( Table 3). Compared with other formulas, the highest value was recorded in fish fed pellet 3 (4 g kg ⁻¹). No significant difference ( P > 0.05) between fish fed 2 and 6 g kg ⁻¹ has been observed ( Table 3). Similarly, in additional groups the activity of index phagocytic (PI), compared with the control of fed fish, was significantly higher ( P < 0.05) ( Table 3). Fish fed dietary pellets 3 showed the highest values ( Table 3). In relation to the activity of respiratory burst (RBA), fish supplemented diets ( P < 0.05) were significantly higher than the control. No significant difference ( P > 0.05) between pellet 2 and pellet 4 was however observed. In comparison to controls after 8 weeks of feeding, and 12 weeks after challenges, significant ( P < 0.05) differences in total leukocyte (TL) activity were observes in fish-feed supplements ( Table 3).

The rate of survival and death and RPS were measured at Week 12 after completion of the challenge test. Unexpectedly, the results showed that treatment with BPE at all concentrations markedly increased the RPS, by >60%, relative to control, although BPE at 4 g kg ⁻¹ provided the maximal protection (99.56%) against A. hydrophila and P. fluorescens infection ( Table 4).

The results of the experiment examining protection against pathogenic bacteria showed a significant increase in goldfish disease resistance, amounting to 68.3–77.0% following all BPE treatments, although no significant difference was measured between the distinct concentrations of BPE ( P > 0.05); moreover, the highest RPS value (77.0%) was obtained with BPE used at 4 g kg ⁻¹ in the fish feed ( Table 4). In conclusion, relative to Pellet 1, which did not contain BPE, all other pellets drastically increased the rate of survival after pathogenic bacterial infection.

No significant differences were present in the quality of water in the goldfish aquaculture media when BPE was included in fish feed. The temperature was set at 29 ± 0.2 °C, the DO was 7.6 ± 0.6 mg L ⁻¹, the pH range was 7.2 ± 0.5, and the total ammonia nitrogen was 0.69 ± 0.24 mg L ⁻¹.

Open Science Framework, OSF 2021: Underlying data for ‘ Boesenbergia pandurata application in goldfish ( Cyprinus carpio) feed to enhance fish growth, immunity, and resistance to bacterial infection’. https://doi.org/10.17605/OSF.IO/827EN. ⁵⁶

This project contains the following underlying data: •

Raw data of the growth Performa (Weight gain)

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).