Manipulation of growth of the Amazonian fish tambaqui, Colossoma macropomum (Characiformes: Serrasalmidae): analysis of physiological and zootechnical aspects

Manipulação do crescimento do peixe amazônico tambaqui, Colossoma macropomum (Characiformes: Serrasalmidae): análises fisiológicas e zootécnicas

ABSTRACT

Colossoma macropomum, known locally as tambaqui, is the native fish most farmed in Brazil, however, technological advances are needed to reach efficient production rates. Modulating growth factors, such as growth hormone, may be associated with improved growth rate and feed efficiency. The use of exogenous hormone for fish rearing is prohibited in Brazil, yet the experimental use of bovine hormone can be useful in research aimed at understanding how to stimulate endogenous growth hormones in fish. Therefore, the present study had the strict objective of understanding the effects of growth hormone on the physiology and zootechnical parameters of C. macropomum under experimental conditions. The animals were intraperitoneally injected every fifteen days with 1, 10 and 100 μg g^-1 of bGH using 0.9% NaCl saline as diluent. The 10 and 100 μg g^-1 bolus had a positive effect on the performance indexes of C. macropomum: weight gain (g), growing length (cm), daily weight gain (g), feed conversion and specific growth rate (% per day). The bGH promoted a greater increase in length than in mass, which caused a reduction in condition factor of the individuals receiving a bolus of 10 and 100 μg g^-1. Furthermore, bGH caused no changes in glucose levels, cortisol, hematological parameters, plasma levels of Na⁺ and K⁺, and activity of gill’s H⁺-ATPase and Na⁺, K⁺-ATPase, at least during the experimental period considered in the present study.

Keywords:
aquaculture; growth performance; osmorregulation; hematological parameters

RESUMO

O tambaqui, Colossoma macropomum, é o peixe nativo mais cultivado no Brasil. No entanto, avanços tecnológicos são necessários para incrementar as taxas de produção. Fatores de crescimento moduladores, como o hormônio do crescimento, podem estar associados a uma melhoria na taxa de crescimento e eficiência alimentar. O uso de hormônio exógeno para a criação de peixes é proibido por lei no Brasil, porém, o uso experimental do hormônio bovino pode ser útil em pesquisas que visam determinar mecanismos de estímulo dos hormônios de crescimento endógenos em peixes. Portanto, o presente estudo teve como objetivo estrito a compreensão dos efeitos do hormônio de crescimento sobre a fisiologia e os parâmetros zootécnicos de C. macropomum em condições experimentais. Os animais foram injetados intraperitonealmente a cada quinze dias com 1, 10 e 100 μg g^-1 de bGH, utilizando solução salina a 0,9% de NaCl como diluente. Observou-se que as concentrações 10 e 100 μg g^-1 tiveram um efeito positivo sobre os índices de desempenho de C. macropomum em ganho de massa (g), crescimento em comprimento (cm), ganho de massa diário (g), conversão alimentar e taxa de crescimento específico (% por dia). O bGH promoveu ganho maior em comprimento do que em massa, o que causou diminuição do fator de condição nos indivíduos que receberam 10 e 100 μg g^-1. Além disso, o bGH não causou alterações nos níveis de glicose, cortisol, parâmetros hematológicos, níveis plasmáticos de Na⁺ e K⁺ e na atividade de H⁺-ATPase e Na⁺, K⁺-ATPase nas brânquias durante o período experimental.

Palavras-chave:
aquicultura; performance em crescimento; osmorregulação; parâmetros hematológicos

INTRODUCTION

Fish consumption will increase as the global human population increases, with aquaculture showing great potential to produce quality food in comparison with traditional fishing activities (FAO 2016). Colossoma macropomum is the most farmed native fish in Brazil (IBGE 2016), however, technological improvements are needed to reach higher efficiency and sustainable production (Bocanegra and Flores 1985; FAO 2016). The expansion of the aquaculture industry has occurred simultaneously with the development of biotechnology. A mix of strategies has been adopted to improve fish growth and meet nutrient requirements, including diets enriched with specific protein nutrients, energy levels and stimulation of endogenous hormones, such as growth hormone (GH) (Li et al. 2003; Hallerman et al. 2007; Reindl and Sheridan 2012; Budi et al. 2015; Vikesa et al. 2017).

The use of exogenous growth hormone in animal husbandry for human consumption is prohibited by current legislation in Brazil, because of possible risks to human health (Sales et al. 2015). However, its use in scientific experimentation is a valuable approach in studies that aim to understand the physiological processes that involve the growth of farm fish, to aid in the development of technologies that improve the production cycle (Peter and Marchant 1995).

The effects of GH on fish include an increase in feed efficiency, appetite, immunity, changes in body composition, behavior, reproductive processes, osmoregulation and tolerance to hypoxia (Li et al. 2003; Canosa et al. 2007; Hallerman et al. 2007). GH can also stimulate lipolysis and fatty acid oxidation in adipose tissue, skeletal and heart musculature and enhance hepatic glycogenolysis, increasing the plasma concentration of glucose (Polakof et al. 2011; Polakof et al. 2012). GH also can interact with cortisol, increasing the capacity of osmotic adjustments (Sakamoto and McCormick 2006). This interaction is associated with increases in plasma metabolite levels, distribution of energy reserves, as well as changes in osmotic pressure and activity of enzymes such as Na⁺, K⁺-ATPase and H⁺ ATPase, which substantially aid in the adaptation of an euryhaline fish to salt water (Liebert and Schreck 2006).

The effects of GH on osmoregulation of freshwater teleosts are almost unknown (McCormick 2001; Arjona et al. 2011), as are the effects of GH on zootechnical performance, hematology of tropical fish, such as C. macropomum. GH has been described to increase growth and metabolism of tropical fish. For example, the bovine GH increased lysozyme activity, plasma immunoglobulin, and growth in Oreochromis niloticus (Leedom et al. 2002; Liñán-Cabello et al. 2013). Possibly the effects caused by exogenous GH occurred due to the high degree of phylogenetic conservation of this molecule, which can present a similarity of 70% to mammalian sequence, considering various species (Peter and Marchant 1995). According to Canosa et al. (2007) and Ma et al. (2012) families of several hormones, including GH, prolactin (PRL), somatotropin, chorionic or placental lactogen (PL) and somatolactin (SL), have arisen from the same common ancestor. For example, the GH of Tinca tinca presents low homology for the structural alignments, the amino acids at the binding sites were similar to human GH (Panicz et al. 2012).

Thus, the aim of this study was to understand the effects of bovine growth hormone (bGH) on aspects of physiology, biochemistry and growth of juveniles of C. macropomum, including hematology, stress markers, and osmorregulation.

MATERIAL AND METHODS

Experimental animals

Juveniles of C. macropomum were purchased from a local fish farmer and transported to the Laboratory of Ecophysiology and Molecular Evolution (LEEM) at the Instituto Nacional de Pesquisas da Amazônia (INPA), where the animals were acclimatized in outdoors tanks supplied with flow-thru low CO₂ water. The Ethics Committee on Animal Experimentation of INPA approved the experimental design and methodology under the registration number 026/2015, and all procedures conformed with Brazilian animal care regulations. The animals were then transferred to their respective experimental units, where they remained for ten days under natural conditions of temperature, photoperiod, and continuous aeration. The levels of dissolved O₂ ranged between 5 and 7 mg L^-1. The fish were fed extruded commercial pellets of 2-4 mm (NUTRIPISCIS®, Brazil) to apparent satiety twice a day, at 8 AM and 5 PM. The elemental composition of the food (measured in a companion laboratory, Fish Nutrition Laboratory at INPA) was 33.6% crude protein, 39.3% carbohydrates, 4% fat, 2.6% crude fiber, 8.7% of dry matter and 11.8% ash.

Experimental procedure

The experiment was conducted under laboratory conditions using 144 fish weighing 51.00 ± 4.24 g and measuring 14.55 ± 0.24 cm total length (mean ± SEM). The animals were randomly assigned to four treatments with three replicates, following a completely randomized design, totaling 12 animals per experimental unit. The bGH (Lactotropin ®; Elanco, Brazil) was dissolved in physiological saline (0.9% NaCl) to a final nominal concentration of 1, 10, and 100 μg g^-1, and was injected intraperitoneally. As control treatment animals were injected only with saline solution. The experimental concentrations were based on Leedom et al. (2002) and Li et al. (2003). Each of the 12 experimental units was composed of a polyethylene tank with a capacity of 150 L, equipped with a biological filter, continuous aeration and a flow rate of 0.5 L min^-1. The experiment lasted 60 days. During this period, fish were weighed and received a bolus of bGH hormone at 0, 15, 30 and 45 days. At each of these timepoints, all animals were transferred to water at ~14°C to reduce their activity and were weighed to the nearest 0.1g using a digital balance, and had the total length (from the beginning of the head to the end of the caudal fin) measured to the nearest 0.1cm using an icthiometer. The bolus of bGH to be injected was based on the measured weight of each animal. For this purpose, three solutions were prepared with the following concentrations: 0.2; 2 and 20 μg μL^-1. These solutions were used in animals injected with 1, 10 and 100 μg g^-1 bGH concentration, respectively. Thus, animals of 50 g received a bolus of 250 μL, using an insulin syringe (1 mL). This procedure was used in order to standardize the amount of injected solution.

Nine fish per treatment, three per tank, were randomly collected for physiological analysis. The animals were euthanized by severing the spinal cord, and gill tissue was collected for the measurement of Na⁺, K⁺-ATPase and H⁺-ATPase activity. Stocking density was adjusted after every biometry to maintain the living mass of 5 g L^-1 of water. Physical and chemical parameters of the water were measured at the beginning and throughout the experimental period, three times a week at 9:00 am (Table 1).

Table 1
Physicochemical characteristics of the water in the experimental units: dissolved oxygen (DO), temperature (Temp), pH, conductivity (Cond), total ammonia and levels of sodium (Na⁺), potassium (K⁺), chloride (Cl^-) and calcium (Ca²⁺). Data presented as mean ± SEM.

Parameter	Control	1 µg g-1	10 µg g-1	100 µg g-1
DO (mg L-1)	5.64 ± 0.32	5.68 ± 0.21	5.51 ± 0.48	5.03 ± 0.35
Temp (ºC)	27.46 ± 1.15	27.49 ± 1.12	27.38 ± 1.10	27.53 ± 1.22
pH	6.25 ± 0.50	6.26 ± 0.52	6.22 ± 0.54	6.21 ± 0.51
Cond (µs cm-1)	110.76 ± 35.65	107.73 ± 35.72	109.39 ± 36.24	112.57 ± 33.89
Ammonia (µM L-1)b	39.50 ± 14.30	43.74 ± 16.79	39.86 ± 14.97	44.01 ± 19.55
Na+ (µM L-1)	140.76 ± 11.49	138.61 ± 17.46	137.60 ± 11.15	146.76 ± 10.44
K+ (µM L-1)	78.51 ± 10.80	77.79 ± 14.45	77.38 ± 7.71	85.37 ± 11.62
Cl- (µM L-1)	73.10 ± 9.44	64.16 ± 14.34	81.47 ± 13.56	76.62 ± 15.33
Ca2+ (µM L-1)	6.19 ± 1.67	5.54 ± 2.04	5.20 ± 1.37	6.38 ± 1.70

• ^b
  Total ammonia (NH3 + NH4⁺).

Growth performance

Daily weight gain (DWG), weight gain, growth in length, feeding efficiency (FE), specific growth rate (SGR), condition factor (CF) and total average individual food consumption (IFC) were determined as follows:

$\mathrm{DWG}(g)\; =\frac{\left[\text{final weight (g)}-\text{initial weight (g)}\right]}{\text{time (days)}}$;

$\text{FE}=\frac{\text{weight gain in the period}(g)}{\text{amount of feed provided}(g)}$;

$\mathrm{SGR}(\text{\% day})\; =\frac{\left[\left(\text{ln final weight}-\text{ln initial weight}\right)\right]}{\text{number of days}}\ast 100$;

$\mathrm{CF}=\mathrm{weight}{\mathrm{length}}^{\text{3}}/$;

$\mathrm{IFC}(g)\; =\frac{\sum \left[\text{amount of feed consumed (g) the period}\right]}{\text{total individuals}}$.

Blood parameters

Nine fish per treatment at 15, 30, 45 and 60 days timepoints were bled from the tail vessel using a 3 mL heparinized (lithium heparin 5.000 IU) syringe. The samples were then stored in 2 mL microtubes (Eppendorf) and were kept on ice. Whole blood was 1:200 diluted in citrate formaldehyde solution (3.8 g of sodium citrate, 2 mL of 40% formalin and distilled water qsp 100 mL) for red blood cell counting under 400X magnification using a Neubauer chamber and an optical microscope Motic Professional B5 (Motic, USA). The hematocrit was determined by the microhematocrit method as described by Goldenfarb et al. (1971). Blood hemoglobin (Hb) concentration was measured by the cyanmethemoglobin method as described by van Kampen and Zijlstra (1961). Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated as described by Brown (1976). Blood samples were centrifuged at 1835 RCF in a Eppendorf 5430R centrifuge (Eppendorf, Germany) to separate the plasma for determination of plasma glucose, cortisol and Na⁺ and K⁺. Plasma glucose was determined using an In Vitro kit (In Vitro Diagnóstico Ltda, Brazil), following manufacturer instructions, and a microplate spectrophotometer Spectramax 384 PLUS (Molecular Devices, USA) as outlined by Bartoňková et al. (2016). Cortisol was determined using a commercial ELISA kit (DBC, Diagnostics Biochem Inc, Canada) according to methods previously outlined by Kelly and Wood (2001). Plasma concentrations of Na⁺ and K⁺ were determined using atomic absorption spectrophotometry (AAnalyst 800, Perkin-Elmer, USA) in flame mode.

Na⁺, K⁺-ATPase and H⁺-ATPase activities

The first gill arch was excised, frozen in liquid nitrogen, and stored at -80 °C before the determination of Na⁺, K⁺-ATPase and H⁺-ATPase activities as described by Kültz and Somero (1995). Frozen gill tissues were homogenized (1:10) in SEID buffer (150 mM sucrose, 50 mM imidazole, 10 mM EDTA, 0.5% Na-deoxycholate, pH 7.5), centrifuged at 4 °C, 4758 RCF in a Eppendorf 5430R centrifuge (Eppendorf, Germany) and the supernatants were saved for enzyme determinations. 5 µL of the supernatant of each sample were added to 12 microplate wells, and were incubated with the reaction solution (30 mM imidazole, 45 mM NaCl, 15 mM KCl, 3 mM MgCl₂·6H₂O, 0.4 mM KCN, 1.0 mM ATP, 0.2 mM NADH, 0.1 mM fructose 1.6 diphosphate, 2 mM phosphoenolpyruvate, 3 IU mL^-1 pyruvate kinase and 2 IU mL^-1 lactate dehydrogenase). Additionally, out of the 12 wells, four received additional 2 mM ouabain and four 2 mM N-ethylmaleimide. The assay is based on inhibition of Na⁺, K⁺-ATPase activity by ouabain and H⁺-ATPase by N-ethylmaleimide. The NADH oxidation rate was read over 10 min at 340 nm at 25^oC. The activity of Na⁺, K⁺-ATPase and H⁺-ATPase was calculated as the difference between total activity to ouabain or to N-ethylmaleimide treated samples, respectively. Protein concentrations of crude homogenates were determined using the Bradford method (Bradford 1976). The activities of Na⁺, K⁺-ATPase and H⁺-ATPase are expressed as micromole ATP mg protein^-1 h^-1.

Statistical analyses

The results are presented as mean ± SEM (standard error of the mean). Fish mass (g) and length (cm) were analyzed and adjusted using the Cochran test (p < 0.05) to ensure homogeneity at the beginning of the experiment. Data normality was checked using Shapiro-Wilk test. For statistical analysis, a two-way ANOVA (time and bGH) was used, followed by a Tukey test comparison to discriminate the significant differences (p < 0.05). One dataset without a normal distribution was analyzed using the non-parametric Kruskal-Wallis test, and the significance of the differences was compared by the Dunn’s test (p < 0.001). A non-linear quadratic exponential regression was performed to determine the behavior of mass gain and length over the 60 experimental days.

RESULTS

A significant weight gain was observed 15 days after initial treatment for the animals injected with 100 μg g^-1 bGH compared to all other experimental conditions (control: p = 0.012; 1 μg g^-1 p = 0.022; 10 μg g^-1 p=0.043). The weight gain persisted over the entire experimental period, and at day 60 the animals injected with 100 μg g^-1 had gained 43.86% in weight in relation to control animals (Figure 1A). Weight gain over the experimental period was described by the following quadratic polynomial equations (Figure 1A): $\text{Control:}Y=-0.0043+0.3963x+0.0048x^2\text{,}{R}^2=0.999\text{; 1}{\text{μg g}}^{-\text{1}}\text{:}Y=0.5735+0.3638x+0.0048x^2\text{,}{R}^{\text{2}}=0.998\text{; 10}{\text{μg g}}^{-\text{1}}\text{:}Y=0.6715+0.306x+0.0089x^2\text{,}{R}^2=0.998\text{; 100}{\text{μg g}}^{-\text{1}}\text{:}Y=0.7592+0.0982x+0.009x^2\text{,}{R}^2=0.997$ .

Figure 1
Effect of intraperitoneal injection of growth hormone (Lactotropin ®) on the growth in weight (A) and length (B) of juvenile tambaqui (Colossoma macropomum) in experimental conditions. Results are expressed as mean ± SEM (n = 3, N = 3). Data points containing a symbol are significantly different from those that do not share the same symbol within the same sampling period (p < 0.05). The quadratic effect of the curves is explained by Y = y0 + ax + bx² where: Y = length in gain and growth in length (cm); x = time (days); y0, and b = coefficients. This figure is in color in the electronic version.

The growth hormone caused a dose-response increase of fish length and weight over time (Length, treatments: F = 27.978, p < 0.001; time: F = 207.13, p < 0.001; interaction F = 5.22, p < 0.001. Weight, treatments: F = 55.99 p < 0.001; time: F = 376.12, p < 0.001; interaction: F = 4.43, p < 0.001). After 45 and 60 days, there was significantly greater growth in length (Figure 1B) of animals injected with 10 and 100 μg g^-1 bGH relative to control animals and to animals injected with 1 μg g^-1. At 60 days the animals that received 10 and 100 μg g^-1 bGH presented an increase of 66.9% and 74.3% in length, respectively, relative to the control. In contrast, animals injected with 1 μg g^-1, did not present a significant growth increase compared to control (Fig. 1B). Growth performance in length over the experimental period was described by the following quadratic polynomial equations (Figure 1B): $\text{Control:}Y=-0.0366+0.0647x+0.0010x^2\text{,}{R}^2=0.999\text{; 1}{\text{μg g}}^{-\text{1}}\text{:}Y=0.1180+0.0326x+0.0018x^2\text{,}{R}^{\text{2}}=0.997\text{; 10}{\text{μg g}}^{-\text{1}}\text{:}Y=0.1474+0.0172x+0.0032x^2\text{,}{R}^2=0.998\text{; 100}{\text{μg g}}^{-\text{1}}\text{:}Y=0.2126+0.0435x+0.0029x^2\text{,}{R}^2=0.997$ .

Daily weight gain (DWG) increased significantly for animals injected with 10 and 100 μg g^-1 over the experimental period (p < 0.001) (Table 2). Feed efficiency (FE) and condition factor (CF) were also improved (p < 0.05) in animals injected with 100 μg g^-1. Average individual food consumption (IFC) increased at day 45 in animals that received 100 μg g^-1 (p < 0.05).

Table 2
Effects of intraperitoneal application of growth hormone (Lactotropin ®) on daily weight gain (DWG), specific growth rate (SGR), feed efficiency (FE), average individual food consumption (IFC) and condition factor (CF) in juvenile tambaqui (Colossoma macropomum) in experimental conditions. Results are shown as mean ± SEM (n = 3, N = 3). Different small letters indicate significant differences among treatments at a given collecting time. Different capital letters indicate differences within a given treatment over time (p < 0.05).

bGH concentration (µg g-1)	DWG* (g)	SGR (% in day)	FE (%)	IFC (g)	CF (%)
	After 15 days
Control	0.63 ± 0.05a	14.63 ± 0.68A	0.63 ± 0.02aA	12.91 ± 0.70A	1.67 ± 0.02aA
1	0.55 ± 0.04a	13.62 ± 0.24A	0.57 ± 0.01a	13.67 ± 0.75A	1.66 ± 0.02a
10	0.59 ± 0.04a	14.50 ± 0.42A	0.68 ± 0.05aAB	13.17 ± 0.94A	1.60 ± 0.02ab
100	0.94 ± 0.05b	17.53 ± 0.76A	0.95 ± 0.02bA	14.92 ± 2.00A	1.56 ± 0.02b
	After 30 days
Control	0.53 ± 0.05a	9.20 ± 0.59B	0.35 ± 0.06aB	16.09 ± 1.75A	1.72 ± 0.03aA
1	0.49 ± 0.03a	8.94 ± 0.41B	0.41 ± 0.03ab	16.73 ± 1.49AB	1.71 ± 0.02a
10	0.55 ± 0.04a	9.35 ± 0.46B	0.45 ± 0.06abA	15.91 ±1.41A	1.63 ± 0.02ab
100	0.80 ± 0.04b	10.66 ± 0.78B	0.62 ± 0.02bB	18.31 ± 2.19AB	1.58 ± 0.02b
	After 45 days
Control	0.64 ± 0.06a	7.43 ± 0.79C	0.59 ± 0.07AB	17.68 ± 0,56aA	1.68 ± 0.02A
1	0.56 ± 0.06a	7.16 ± 0.94C	0.67 ± 0.14	16.59 ± 1.15aAB	1.66 ± 0.02
10	0.69 ± 0.06ab	7.63 ± 0.59C	0.78 ± 0.06B	17.85 ± 0.50aA	1.61 ± 0.02
100	0.92 ± 0.05b	8.26 ± 0.55CD	0.78 ± 0.11AB	22.21 ± 1.49bAB	1.61 ± 0.03
	After 60 days
Control	0.76 ± 0,11ab	6.73 ± 0.66D	0.63 ± 0.05A	27.05 ± 3.15B	1.86 ± 007aB
1	0.66 ± 0.08a	6.30 ± 0.69D	0.62 ± 0.04	21.27 ± 1.75B	1.75 ± 0.02ab
10	1.01 ± 0.13b	6.64 ± 0.59D	0.65 ± 0.12AB	27.87 ± 5.93B	1.68 ± 0.02b
100	1.09 ± 0.06b	6.88 ± 0.41D	0.82 ± 0.04AB	30.54 ± 5.51B	1.68 ± 0.03b

• *DWG data were analyzed by Kruskal-Wallis test, and the differences were compared using Dunn’s test (p < 0.001). SGR, FE, TIR and CF were analyzed by ANOVA, followed by Tukey post hoc test (p < 0.05)

Except for decreased hematocrit in animals injected with 10 and 100 μg g^-1 at day 60, compared to control and 1 μg g^-1 (p = 0.045 and p = 0.028, respectively), no changes were observed for all other hematological parameters (Table 3). Cortisol levels showed no difference (F = 0.209, p = 0.933) among treatments at any sampling time, except for a decrease observed at day 15 (p < 0.001 for all comparisons) (Figure 2A). Likewise, except for the variation observed in control animals over the whole experimental period, plasma glucose levels showed no changes among the experimental treatments (Figure 2B).

The activity of Na⁺, K⁺-ATPase was reduced over time in all treatments, except for control animals (Figure 3A). In contrast, no differences in the activities of H⁺-ATPase were observed over the whole experimental period (Figure 3B). The lowest values for plasma levels of sodium (Na⁺) were observed at day 45, while no changes among treatments over time were observed for potassium levels (K⁺) (Figure 4).

Table 3
Effects of intraperitoneal application of growth hormone (Lactotropi n ®) on hematocrit (Ht), blood hemoglobin (Hb), red blood cells (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) in juvenile tambaqui (Colossoma macropomum) in experimental conditions. Results are shown as mean ± SEM (n= 3, N= 3). Different letters indicate significant diff-erences among treatments at a given time point (p < 0.05).

Concentration (µg g-1)	Ht (%)	Hb (g dL-1)	RBC (nºerit.106/mm3)	MCV (µm³)	MCH (pg)	MCHC (%)
	After 15 days
Control	30.06 ± 0.96	7.88 ± 0.27	1.64 ± 0.07	184.76 ± 7.22	48.69 ± 2.79	26.31 ± 0.76
1	29.97 ± 1.15	8.04 ± 0.33	1.67 ± 0.06	179.71 ± 4.96	48.38 ± 2.22	26.89 ± 0.81
10	29.94 ± 0.70	8.23 ± 0.20	1.85 ± 0.06	162.78 ± 4.42	44.69 ± 0.82	27.53 ± 0.52
100	26.00 ± 0.95	7.52 ± 0.23	1.56 ± 0.06	168.70 ± 9.13	48.64 ± 2.04	28.97 ± 0.42
	After 30 days
Control	27.88 ± 1.10	7.96 ± 0.31	1.85 ± 0.14	156.76 ± 10.52	43.99 ± 1.55	28.91 ± 1.69
1	29.16 ± 1.42	8.12 ± 0.29	1.83 ± 0.10	161.78 ± 8.55	45.05 ± 1.93	28.02 ± 0.58
10	29.27 ± 0.99	8.41 ± 0.37	1.85 ± 0.07	159.02 ± 3.97	45.93 ± 2.48	28.77 ± 0.98
100	26.50 ± 0.93	7.12 ± 0.31	1.72 ± 0.08	154.45 ± 5.19	41.50 ± 1.57	26.88 ± 0.60
	After 45 days
Control	29.11 ± 0.6	7.47 ± 0.21	1.49 ± 0.07	197.86 ± 8.01	50.64 ± 1.78	25.68 ± 0.61
1	28.61 ± 1.40	7.43 ± 0.45	1.59 ± 0.09	181.92 ± 6.97	46.98 ± 1.54	25.97 ± 0.88
10	28.44 ± 0.84	7.72 ± 0.31	1.58 ± 0.10	183.20 ± 8.27	49.48 ± 1.80	27.17 ± 0.78
100	27.37 ± 1.68	7.29 ± 0.54	1.47 ± 0.11	188.59 ± 8.86	49.71 ± 1.90	26.61 ± 1.15
	After 60 days
Control	33.55 ± 1.00b	9.03 ± 0.33	1.82 ± 0.13	193.35 ± 16.65	51.17 ± 3.11	27.10 ± 1.32
1	31 ± 0.68ab	8.77 ± 0.24	1.81 ± 0.10	174.19 ± 8.01	49.46 ± 2.72	28.37 ± 0.87
10	29.44 ± 1.14a	8.85 ± 0.63	1.64 ± 0.06	180.58 ± 7.41	54.28 ± 3.95	29.97 ± 1.58
100	29.16 ± 0.70a	8.99 ± 0.39	1.85 ± 0.11	162.04 ± 10.94	49.52 ± 2.89	30.84 ± 1.05

Figure 2
Effect of intraperitoneal injection of growth hormone (Lactotropin ®) on plasma concentrations of cortisol (A) and glucose (B) in juvenile tambaqui (Colossoma macropomum) in experimental conditions. Results are expressed as mean ± SEM (n = 3, N = 3). Different letters indicate significant difference for a treatment throughout the sampling period (p < 0.05). This figure is in color in the electronic version.

Figure 3
Effect of intraperitoneal injection of growth hormone (Lactotropin ®) on activity of Na⁺, K⁺-ATPase (A) and H⁺-ATPase (B) in juvenile tambaqui (Colossoma macropomum) in experimental conditions. Results are expressed as mean ± SEM (n = 3, N = 3). Different letters indicate significant difference for a given treatment thoughout the sampling period (p < 0.05). This figure is in color in the electronic version.

Figure 4
Effect of intraperitoneal injection of growth hormone (Lactotropin ®) on plasma concentrations of Na⁺ (A) and K⁺ (B) in juvenile tambaqui (Colossoma macropomum) in experimental conditions. Results are expressed as mean ± SEM (n = 3, N = 3). Different letters indicate significant difference for a given treatment throughout the sampling period (p < 0.05). This figure is in color in the electronic version.

DISCUSSION

The use of intraperitoneal injection of bovine growth hormone to improve growth of C. macropomum juveniles was shown to be effective, as the administration of bGH at the two highest doses (10 and 100 μg g^-1) caused an increase in weight, length, specific growth rate and daily weight gain. Similar effects have been described for other farmed fish species, such as Scaphirhynchus platorhynchus, Sparus aurata, Oncorhynchus mykiss, Oreochromis niloticus and Paralichthys olivaceus (Cavari et al. 1993; Moriyama et al. 1993; Leedom et al. 2002; Li et al. 2003; Liu et al. 2012; Fenn and Small 2015). The growth improvement is likely to be due to the direct effect of GH up regulating protein synthesis, and to the indirect effect on cell metabolism. In muscle and liver, for example, GH stimulates the release of insulin release factors (Leggatt et al. 2009). Among these factors is IGF-1 (insulin growth factor-I) which acts as a regulator of amino acid transport to the tissues, and as a signal for cell division (Hossner 2005; Takei and Loretz 2006; Reindl and Sheridan 2012).

Liñán-Cabello et al. (2013) suggested that growth may be promoted by increased amino acid transport into the cell membrane and by inducing the release of fatty acids from adipose tissue, increasing the concentration of free fatty acids in body fluids. With an increased flow of fatty acids, the tissues are stimulated to convert them into acetyl-CoA, which is subsequently used as a source of energy, saving protein for growth (Bjornsson et al. 2002; Hossner 2005). However, there were no differences in blood glucose among treatments in this study, and control animals had greater variation range of glucose over time than treatment animals. In this sense, Sangiao-Alvarellos et al. (2005) showed that injection of 2 and 5 μg g^-1 of sheep growth hormone increased glycogenolys potential in the gills, kidneys, and brain of Oncorhynchus mykiss. Leung et al. (1991) also found that 50 and 100 μg g^-1 bGH injected in Oreochromis mossambicus caused a substantial reduction in liver glycogen and a decrease in the activity of glycogen synthase, resulting in higher plasma levels of glucose with simultaneously increased levels of amino acids. Thus, having more energy available, the larger part of the food intake could be directed to somatic growth (Bjornsson et al. 2002). Therefore, the higher growth in length of C. macropomum after 45 days receiving 10 and 100 μg g^-1 bGH, but only for animals receiving 100 μg g^-1 for growth in mass, may be the result of greater bone growth relative to muscle growth, and lipolysis in adipose tissue. Likewise, Oreochromis aureus injected with bGH for seven weeks presented higher lengths, probably because exogenous GH has little influence on the production of endogenous GH, which maximizes the influence of GH on growth of bone cartilage, thus impacting skeletal form (Wille et al. 2002).

The condition factor of animals reflected the increase in length of C. macropomum. Administration of bGH also promoted a decrease in the condition factor of Ictalurus punctatus (Peterson et al. 2004), of juveniles of Saxatilis morone (Hunt et al. 2000) and Oreochromis niloticus (Liñán-Cabello et al. 2013). The reasons for the stimulation of growth in length rather than in mass by bGH are unclear, and more studies are needed to explain this phenomenon. It may, however, be related to the allometric relations in tissue growth, since bone tissue has a natural priority over muscle tissue during the growth phase among vertebrates (Hossner 2005). Yet, growth in mass was also reported for fish receiving GH. Anguilla japonica injected with a bolus of GH showed an increased incorporation of food leucine [¹⁴C] in muscle and liver, reflecting increased food intake and greater growth in mass (Inui and Ishioka 1985). Furthermore, the use of GH increased protein retention and reduced the level of lipids in fish (Macari et al. 1994; Fenn and Small 2015). Improved feed conversion and higher growth was also reported for animals receiving recombinant bovine growth hormone (Silverstein et al. 2000; Liñán-Cabello et al. 2013).

It should be also considered that anabolic hormones like GH, prolactin and IGFs affect the immune system, where the axis GH/IGF-1 plays a key role (Hooghe et al. 1993). By controlling the size of bones and organs of the animal, the GH, and IGF-1 also control, though indirectly, the hematopoietic centers in the bone marrow, increasing the production of blood cells (Clark 1997). Thus, erythropoiesis can be related to the GH affinity to prolactin and erythropoietin (Epo) (Moritz et al. 1997). Salmon (Oncorhynchus kisutch) receiving growth gene OnMTGH1 had a significant increase in hematocrit (%), blood hemoglobin (g dL^-1), MCH, MCV and MCHC, and significantly decreased the number of white cells (Kim et al. 2013). In our study, however, only at day 60 did hematocrit decrease in animals injected with bGH, suggesting that bGH did not promote changes in the hematological condition of the animals. Similarly, Li et al. (2011) found no changes in hematocrit, hemoglobin, and MCHC of carp (Cyprinus carpio L.) microinjected with recombinant GH (pCAgcGH, transgenic gene) and subjected to exercise to exhaustion.

The growth hormone also seems to play an important role in osmoregulation and smoltification of fish that travel between different environments (Reinecke 2010). GH acts on the acclimatization process to salt water, while prolactin acts on the acclimatization to freshwater, and cortisol interacts with both hormones facilitating the process (McCormick 2001; Sakamoto and McCormick 2006). More studies are needed to clarify the effects of GH on osmoregulation of tropical freshwater teleosts, particularly for those living in ion-poor waters, as is the case of C. macropomum.

Several studies have described increased activity of Na⁺, K⁺ -ATPase in chloride cells and increased plasma osmolality in GH injected animals (Madsen et al. 1994; Arnesen et al. 2003; Handeland et al. 2003; Sangiao-Alvarellos et al. 2005). However, we observed no changes in cortisol, and in the activity of Na⁺, K⁺-ATPase and H⁺-ATPase, nor in plasma concentration of sodium and potassium in C. macropomum injected with bGH. As the analyzed animals were not subjected to an osmoregulatory challenge, there was no need for further regulation of Na⁺, K⁺-ATPase activity. The studies describing GH effects on Na⁺, K⁺-ATPase activity were carried out on animals subjected to sudden changes in water salinity (Mancera and McCormick 1998; Arnesen et al. 2003; Liebert and Schreck 2006. More research is needed to shed light on the regulation mechanisms of Na⁺, K⁺-ATPase activity in tropical freshwater fish.

CONCLUSIONS

In a 60-day experimental study, the growth of juvenile C. macropomum was positively influenced by bGH without detectable effects on health and osmoregulation. It is concluded that bGH may be a valuable tool in scientific studies directed towards growth manipulation. As administration to C. macropomum caused an increase in yield, further studies should focus on viable strategies that stimulate the endogenous release of GH in fish. Yet again we stress that the use of exogenous growth hormone in animal production is prohibited in Brazil, and that this study does not recommend the use of bGH in commercial C. macropomum aquaculture

ACKNOWLEDGMENTS

This study was supported by INCT ADAPTA (FAPEAM/CNPq/CAPES). The authors acknowledge the support of Secretaria de produção rural do Amazonas - SEPROR/AM for fish supply and Maria de Nazaré Paula da Silva, Dulcilene Martins, Carolina Abrahim, Renan Amanajás, Rúbia Maielli, Grazyelle Sebrensky and Ivã Guidini for the help in carrying out the experiment. ALP and ALV were recipients, respectively, of a MSc fellowship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and a research fellowship from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).

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• CITE AS:

  Paz, A. L.; Val, A. L. 2018. Manipulation of growth of the Amazonian fish tambaqui, Colossoma macropomum (Characiformes: Serrasalmidae): analysis of physiological and zootechnical aspects. Acta Amazonica 48: 197-206

Edited by

ASSOCIATE EDITOR:

Rodrigo do Valle

Publication Dates

• Publication in this collection
  Jul-Sep 2018

History

• Received
  18 Jan 2018
• Accepted
  25 Apr 2018