Effects of Curcuma longa rhizome on growth , skin pigmentation , and stress tolerance after transport of Trichogaster labiosa

We aimed to evaluate the effects of Curcuma longa as growth promoter, skin pigmentation enhancer, and stress reducer in diets of Trichogaster labiosa after transport. We used five diets containing 0.0, 1.0, 5.0, 10.0, and 25.0 g kg−1 of turmeric rhizome powder. We observed quadratic effects of turmeric supplementation for feed intake, weight gain, final length, and specific growth rate. The estimated amount of turmeric that decreased these variables ranged from 15.53 to 16.39 g kg−1. Quadratic effects of supplementation of turmeric for cyan and black dorsal skin coloring indices were observed, with estimated values that increased these variables equal to 15.03 and 17.44 g kg−1, respectively. After fish transport, quadratic effects of turmeric were observed for the cyan and black dorsal skin depigmentation indices, with estimated values that increased these variables equal to 13.29 and 17.04 g kg−1, respectively. These results demonstrate that supplementation with turmeric at levels up to 17 g kg−1 causes further reduction in skin color due to the stress of transport. Thus, Curcuma longa acts neither as a growth promoter nor as a stress reducer for Trichogaster labiosa. Curcuma longa does not improve the orange pattern of skin pigmentation in the strain of T. labiosa orange thick-lipped gourami.


Introduction
In ornamental fish trade, animals experience procedures, such as capture, size sorting, and fasting before transport, which can trigger stress responses and cause a decrease in skin color and consequent reduction of its market value (Dharmaraj and Dhevendaran, 2011).In fish that remain in ponds, frequent captures and size-sorting management can cause chronic stress responses once fishing is partial.Under these conditions, the fish have reduced growth (Barton, 2002), decreased activity of their immune systems (Wendelaar Bonga, 1997), and lower disease resistance (Lima et al., 2006).
To reduce losses associated with stress, it is common to use additives in the water, such as sodium chloride (Gomes et al., 2003;Brandão et al., 2008) and anesthetics (Becker et al., 2016;Moreira et al., 2015).However, the use of additives in fish diets in preparation for transport has not been well studied.The use of these additives can help increase stress resistance (Aly and Mohamed, 2010;Zeppenfeld et al., 2014) and improve the activity of the immune system (Rao et al., 2006;Sahu et al., 2008).
The Trichogaster genus is among the most popular ornamental fish species in the world (Rüber et al., 2006).Trichogaster labiosa, formerly classified as Colisa labiosa, stands out due to its docility and peaceful coexistence with other species.The strain known as "orange thick-lipped gourami" is widely marketed due to its orange coloration.This strain resulted from genetic improvements that replaced the barred blue and orange patterns to a uniformly orange pattern.
In this study, we aimed to evaluate the potential of C. longa as growth promoter, skin pigmentation enhancer, and stress reducer for T. labiosa after transport.
The experiment consisted of a randomized block design with five treatments and two replicates within each block, totaling 20 experimental units.Block 1 (B1) consisted of fish with an average weight of 0.61±0.02g, and block 2 (B2) contained fish with an average weight of 0.77±0.02g.The treatments consisted of five practical diets containing 0.0, 1.0, 5.0, 10.0, and 25.0 g kg −1 of turmeric rhizome (C.longa) powder (Table 1).Each aquarium was considered a replicate.Effects of Curcuma longa rhizome on growth, skin pigmentation, and stress tolerance after... Nascimento et al.

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The experimental diets were formulated based on the nutritional requirements of Trichogaster lalius (Zuanon et al., 2013) (Table 1) and chemical composition of turmeric rhizome powder (Table 2).The turmeric rhizome powder (Açafrão da Terra Pirata ® -Contagem, Minas Gerais, Brazil) was premixed with the other ingredients and then pelletized, dried in a forced-ventilation oven (40 °C for 48 h), ground, and sieved to obtain pellets of 1±0.2 mm diameter.
Orange thick-lipped gourami, T. labiosa, juveniles were kept in 7-L aquaria, which were provided with aeration and biological filters, at a stocking density of 1.71 fish L −1 of water (12 fish aquarium −1 ).
Temperature was controlled by heaters and thermostats (26.3±0.15°C).Fish were fed to satiation three times daily for 120 d.The water temperature was checked daily at 08:00 h.Dissolved oxygen was measured biweekly using an oximeter.The pH, total ammonia, and nitrite were measured using colorimetric kits.Conductivity was measured with a conductivity meter.Toxic ammonia was calculated using the formula: Toxic ammonia = Total ammonia After verification of these water quality parameters, aquaria water was siphoned to remove any feces.
At the end of the experimental period, fish were counted, weighed, and measured using the following standard measurements for calculating these growth performance variables: survival rate, weight gain, feed intake, feed conversion ratio, protein efficiency ratio, specific growth rate, and body condition factor.
To evaluate fish skin pigmentation, four fish per aquarium (two males and two females) were euthanized by high amounts of anesthetic (400 mg L −1 clove oil), dried on paper napkins, and imaged according to the methodology of Rezende et al. (2012) with modifications.The images were collected in RAW format with a Panasonic DMC-FZ200 digital camera.Fish were placed individually on a white background (A4 sheet paper over a Styrofoam base) next to an 18% grey card (medium grey).A luminaire with a fluorescent bulb (45 W) was positioned 35 cm above the surface on which the fish were placed, forming a 90° angle with the axis of the animal body.To enhance the lighting and homogenize the light distribution, a Styrofoam base was positioned 15 cm from the belly of the animal, forming a 90° angle with the Styrofoam base.To obtain maximum fidelity to the actual colors of the fish, the images in RAW format were treated in a standardized manner following the technique of balancing colors from the 18% grey card (medium grey) with Adobe Lightroom ® software.As the RAW format decomposes the colors in the RGB system (red, blue, and green), digital files were converted to CMYK format (C, cyan; M, magenta; Y, yellow; and K, black) to allow assessment of the color of interest in this study (ranging from yellow to magenta, according to the pigment present in turmeric).These levels of skin coloration were measured in two body parts (ventral and dorsal) of males and females.In each body part, two images (replicas) were taken to calculate the average values for each color index (Figure 1).
To calculate the gonadosomatic and viscerosomatic indices, we used the same fish that were euthanized for evaluation of skin pigmentation (two males and two females per aquarium).The viscera and gonads were subsequently removed and weighed on an analytical balance.
After evaluation of growth performance and skin color, the remaining fish were returned to the aquaria to recover from the stress of capture and handling biometrics and were fed the same test diets for another 55 d (totaling a period of 175 d of feeding).After this period, fish fasted for 48 h, after which they were placed in 15 plastic bags (five treatments and three replicates) with dimensions of 35.5 × 25 cm.Each bag was considered a replicate and contained 1 L of water (8 fish L −1 ), approximately 1 L of atmospheric air, and approximately 1 L of oxygen introduced by a hose coupled to an oxygen cylinder.
Fish remained in plastic bags for 24 h, following the methodology described by Teo et al. (1989).The bags with the fish were kept in the trunk of a car, which alternated between periods of motion and no motion.After this period, bags were opened, and water samples were taken for the following analyses: dissolved oxygen, temperature, pH, and total and toxic ammonia.
To measure blood glucose and lactate levels, two fish per bag (six fish per treatment) were euthanized by high amounts of anesthetic (400 mg L −1 clove oil).The caudal peduncle was subsequently cut with a scalpel, and blood was directly deposited on a reagent strip of a digital monitor device (Accutrend ® Plus Roche -Mannheim, Germany).
To evaluate the stress effect of transport on the skin pigmentation changes, two males and two females per bag (12 fish per treatment) were imaged, and skin-coloring indices (SCI) were evaluated following the same procedures described earlier.Based on the coloring indices, skin depigmentation indices (SDI) were calculated using the following expression: in which SCI BT = skin coloring index before transport (%), and SCI AT = skin coloring index after transport (%).
Evaluations of the effects of turmeric levels in the diet on growth performance, body indices, and water quality before and after transport and stress responses after transport (blood glucose and lactate) were performed by analysis of variance (ANOVA) and polynomial regression at 5% probability.The Lilliefors test was applied to check the assumption of normality of errors.To check the homogeneity of error variances among treatments, Bartlett's test was applied.To choose regression models, we considered the significance of the regression coefficients, behavior of the variables under study, and magnitude of the correlation coefficients, calculated according to the regression sum of squares/treatments sum of squares.
The evaluations of turmeric in the diet and sex effects on the skin coloring indices and skin depigmentation indices were performed by a two-way ANOVA.When the F test was significant at 5% probability, polynomial regression was performed.Data were analyzed using SAEG (Sistema para Análises Estatísticas, version 9.1) statistical software package.

Results
No effects of turmeric supplementation in the diet were observed on survival rate, feed conversion ratio, body condition factor, viscerosomatic index, and male and female gonadosomatic indices (P>0.05)(Table 3).Quadratic effects of dietary turmeric for weight gain, final length, specific growth rate, and feed intake (P<0.05) were observed, with estimated values for minimizing the variables equal to 15.53, 16.39, 14.94, and 16.21 g kg −1 of diet, respectively (Table 3).For dissolved oxygen (P<0.05) in aquaria water, a quadratic effect of dietary turmeric in the diet was observed, with the estimated value for maximizing this variable equal to 1.39 g kg −1 (Table 4).No significant interactions were observed between the effects of turmeric and sex on skin coloring (P>0.05).No effects of dietary turmeric were observed in the skin coloring indices of cyan ventral (SCI CV ), magenta ventral (SCI MV ), yellow ventral (SCI YV ), black ventral (SCI KV ), magenta dorsal (SCI MD ), and yellow dorsal (SCI YD ) for T. labiosa (P>0.05).For the cyan dorsal (SCI CD ) and black dorsal (SCI KD ), quadratic effects (P<0.05) of dietary turmeric in the diet were observed, with the estimated values that maximize these variables equal to 15.03 and 17.44 g kg −1 of turmeric, respectively (Table 5).No effects of sex for dorsal coloring indices (P>0.05) were observed.Males had higher values of SCI CV , SCI MV , SCI YV , and SCI KV compared with females (Table 5).
After simulation of transport, fish mortality was not observed in any treatment.No significant interactions (P>0.05) were observed between the effects of the turmeric and sex on the skin depigmentation indexes.There were no effects of turmeric supplementation on magenta ventral (SDIM MV ), black ventral (SDI KV ), magenta dorsal (SDI MD ), and yellow dorsal (SDI YD ) skin depigmentation indexes (P>0.05) of T. labiosa.For the cyan dorsal (SDI CD ) and black dorsal (SDI KD ) skin depigmentation indices, quadratic effects (P<0.05) of dietary turmeric were observed, with the estimated values for maximizing the respective variables equal to 13.29 and 17.04 g kg −1 (Table 6).CV -coefficient of variation.ns -not significant for levels of turmeric diet by analysis of variance at 5% probability.
L and Q -linear and quadratic effects, concerning the inclusion of turmeric in the diet.
Effects of Curcuma longa rhizome on growth, skin pigmentation, and stress tolerance after... Nascimento et al. 7 After the transport simulation, quadratic effects of dietary turmeric were observed only on water pH (P<0.05) in plastic bags, with the estimated value for maximizing this variable equal to 16.99 g kg −1 diet (Table 7).
No effects of turmeric supplementation on concentrations of blood lactate and glucose (P>0.05) after transport simulation were observed (Table 8).

Discussion
The growth reduction in T. labiosa fed diets supplemented with turmeric probably occurred due to lower feed intake.Sambaiah et al. (1982) attributed the reduction of feed intake of rats to the low palatability of turmeric.Kang et al. (2011) observed that curcumin, the main constituent of turmeric, reduced feed intake of goldfish, Carassius auratus, probably by inducing the release of corticotrophinreleasing hormone (CRH), a potent anorexigenic neuropeptide (Matsuda, 2009).In laying hens, turmeric also reduced food intake and weight gain (Qasem et al., 2015).Despite the reduction in growth of T. labiosa, supplementation with turmeric did not affect the weight gain of the Nile tilapia (Oreochromis niloticus; Mahmoud et al., 2014) and improved the growth performance of guppy (Poecilia reticulata; Mukherjee et al., 2009).
The reduction in oxygen content of the water in aquaria indicates that turmeric caused an increase in fish metabolism.It is possible that turmeric acted on the lipid metabolism of animals, increasing their oxidation and, thus, increasing oxygen consumption by the fish.Reduced levels of triglycerides and lowdensity lipoprotein cholesterol have been previously demonstrated in laying hens (Riasi et al., 2012), rats (Kim and Kim, 2010), and hamsters (Jang et al., 2008) fed diets containing turmeric or curcumin.Mahmoud et al. (2014) also found reduced total lipids in carcasses of Nile tilapia fed diets with turmeric.
The increased oxidation and decreased fatty acid synthesis in the liver of hamsters was demonstrated by Jang et al. (2008), with reduced fatty acid synthase activity and increased fatty β oxidation activity.Similar results were observed by Ejaz et al. (2009) with increased carnitine palmitoyltransferase-1 expression and reduction of glycerol-3-phosphate acyl transferase-1 activity.However, turmeric did not cause a reduction in dissolved oxygen content in the water in plastic bags after transportation.This may be related to the injection of oxygen into the packages before transportation.Thus, even though  L and Q -linear and quadratic effects, concerning the inclusion of turmeric in the diet.
the turmeric-fed fish may have consumed more oxygen, this effect was not observed because the water was saturated with oxygen.
Because the pigments present in turmeric are yellow-orange, an effect of turmeric supplementation in cyan color was not expected.However, the wild variety of this species shows a pattern of staining of orange and cyan vertical bars.Thus, as turmeric was not efficient in orange pigmentation, it is possible that in the absence of proper pigmentation of orange chromatophores (xanthophores and erythrophores), the underlying chromatophores with cyan pigments (cyanophores) stand out.This phenomenon, known as blue color syndrome, has been observed in Penaeus monodon shrimp, which normally have greenish-brown color, but, under conditions of carotenoid deficiency, they exhibit blue staining (Howell and Matthews, 1991).The increase of the black dorsal coloring index (SCI KD ) shows that the fish were darker, probably due to increased melanin in melanophores and/or dispersion of pigment granules.This effect was probably caused by curcumin, since Jang et al. (2008) demonstrated that this substance regulates proteins that modulate melanogenesis in B16F10 mouse melanoma cells.
Although turmeric did not promote skin pigmentation in T. labiosa, Mukherjee et al. (2009) reported that turmeric powder at a concentration of 0.9 g kg −1 of feed resulted in the highest pigment concentration in caudal fin and muscle of P. reticulata.
Although there was no significant effect of the interaction between curcuma and sex, differences between males and females were observed in cyan, magenta, yellow, and black skin coloring indices of the ventral region.As many species of fish exhibit different color patterns between males and females (generally, males are the more colorful), this result was already expected.Skin color is an important feature in interactions among fish, since it signals social status.In addition, in males, the most intense pigmentation makes them more attractive to females (Rodrigues et al., 2009).The preference of females for males with more intense red coloration of the skin was observed in gourami (Colisa lalia) (currently classified as Trichogaster lalius; Baron et al., 2008), guppy (Houde, 1997), Gasterosteus aculeatus (McLennan and McPhail, 1990), and Betta splendens (Clotfelter et al., 2007).
Although some studies have shown that turmeric attenuates stress responses in mammals with reduction of serum levels of CRH or cortisol (Xia et al., 2006;Xia et al., 2007;Wei et al. 2010), our results indicated that turmeric did not influence the hypothalamus-pituitary-interrenal axis of T. labiosa and, therefore, does not influence the values of blood glucose and lactate.However, some studies have shown that curcumin stimulates the adrenal glands to secrete cortisol or cortisone in mammals (Srivastava and Srimal, 1985;Enyeart et al., 2008).The different effects of curcuma/curcumin on stress control, in different studies, may be related to the curcumin concentrations in different turmeric sources used.
The stress caused by transport frequently induces skin color reduction in ornamental fish (Dharmaraj and Dhevendaran, 2011).In the present study, the supplementation with turmeric at levels up to 17 g kg −1 caused greater reduction in skin color of T. labiosa, even with no increase in stress responses (glucose and lactate).Thus, turmeric may cause reduction in skin color by another way, such as the α-MSH signal pathway.Jang et al. (2009) observed that curcumin reduced the melanin content in B16F10 cells.The referred study shows that hypopigmentation caused by turmeric extract occurred by suppression of melanin synthesis mediated by the activation of the intracellular signaling pathways mitogen-activated protein kinase⁄extracellular signal-regulated kinase and phosphatidylinositol 3-kinase⁄Akt.The activation of this signal pathway caused the suppression of microphthalmia-associated transcription factor expression, including the tyrosinase and tyrosinase-related proteins.

Conclusions
Curcuma longa does not act as a growth promoter or a stress reducer for Trichogaster labiosa and does not improve the orange pattern of the skin pigmentation in the orange thick-lipped gourami strain of T. labiosa.
Effects of Curcuma longa rhizome on growth, skin pigmentation, and stress tolerance after... Nascimento et al.
The levels of skin coloration (C, cyan; M, magenta; Y, yellow; and K, black) were measured in ventral (V) and dorsal (D) regions of females (A) and males (B).

Figure 1 -
Figure 1 -Color evaluation of the body parts of Trichogaster labiosa.

Table 3 -
Productive performance of Trichogaster labiosa fed diets containing increasing levels of Curcuma longa

Table 4 -
Water quality of Trichogaster labiosa fed diets containing increasing levels of Curcuma longa CV -coefficient of variation.ns-not significant by analysis of variance at 5% probability.L and Q -linear and quadratic effects, concerning the inclusion of turmeric in the diet.1 O 2 = −0.0009x 2 − 0.0025x + 6.2595; R 2 = 0.82.

Table 5 -
Skin coloring indexes in the CMYK (C, cyan; M, magenta; Y, yellow; and K, black) patterns of the ventral and dorsal regions of males and females of Trichogaster labiosa fed diets containing increasing levels of Curcuma longa SCI VC -skin coloring index of ventral cyan; SCI VM -skin coloring index ventral magenta; SCI VY -skin coloring index ventral yellow; SCI VK -skin coloring index ventral black; SCI DC -skin coloring index dorsal cyan; SCI DM -skin coloring index dorsal magenta; SCI DY -skin coloring index dorsal yellow; and SCI DK -skin coloring index dorsal black; CV -coefficient of variation.ns -not significant by analysis of variance at 5% probability. 1 SCI DC = −0.0497x 2 + 1.494x + 42.115; R 2 = 0.84. 2 SCI DK = −0.0772x 2 + 2.6935x + 35.183;R 2 = 0.84.

Table 6 -
Skin depigmentation indexes in the CMYK (C, cyan; M, magenta; Y, yellow; and K, black) patterns of the ventral and dorsal regions of males and females of Trichogaster labiosa fed diets containing increasing levels of Curcuma longa, after transport SDI VC -skin depigmentation index of ventral cyan; SDI VM -skin depigmentation index ventral magenta; SDI VY -skin depigmentation index ventral yellow; SDI VK -skin depigmentation index ventral black; SDI DC -skin depigmentation index dorsal cyan; SDI DM -skin depigmentation index dorsal magenta; SDI DY -skin depigmentation index dorsal yellow; and SDI DK -skin depigmentation index dorsal black; CV -coefficient of variation.

Table 8 -
Blood lactate and glucose levels of Trichogaster labiosa fed diets containing increasing levels of Curcuma longa

Table 7 -
Water quality after transport of Trichogaster labiosa fed diets containing increasing levels of Curcuma longa