Influence of garlic extract on larval performance and survival of juvenile angelfish Pterophyllum scalare during transport

Abstract The objective of this study was to evaluate the properties of garlic as a growth promoter in larvae of angelfish Pterophyllum scalare and its benefits during transport of juveniles of this species. The experiment was conducted in recirculation aquaculture system, consisting of 20 tanks of 40 L equipped with independent water input and output. We used 1,400 larvae distributed among five treatments, with four repetitions, which totaled 20 experimental units. The treatments were 0, 50, 100, 200, and 400 mg of garlic extract per kilo of feed. The results showed that the inclusion of levels of garlic extract in the feed did not significantly effect the fish’s development or their transport. Neither did the inclusion of levels of garlic extract affect the survival of the larvae during the trial period. New research with extracts of higher dosages should be performed to elucidate the effect of garlic extract as a growth promoter.


Introducion
The ornamental fish trade is globally recognized, and is in great expansion in Brazil (Ladislau et al., 2019;Oliveira et al., 2016). The inclusion of new species in the market, together with improvements in production techniques and implementation of new technologies have contributed to market growth. One of these ornamental fish, which is caught in the wild and produced in captivity, is the angelfish (Pterophyllum scalare). Angelfish can be produced in captivity in different cultivation systems and may be worth up to 10 times more than the specimens caught in the wild. There are several varieties of colors, such as koi, marble, gold, smokey, black lace, and leopard (Ribeiro et al., 2009).
For ornamental fish, the same nutritional requirements are necessary as those provided when rearing fish for human consumption, and may only have the addition of some carotenoids for use in skin pigmentation. However, ornamental fish are subject to much more stressors during marketing than fish for consumption. These stressors are related to capture, packaging, transport and limnological changes arising from these processes, which can contribute to significant losses throughout O estudo teve como objetivo avaliar as propriedades do alho como promotor de crescimento em larvas de acará bandeira Pterophyllum scalare e seus benefícios no transporte de juvenis da mesma espécie. O experimento foi conduzido em sistema de recirculação composto por 20 aquários de 40 L de volume útil, dotado de entrada e saída de água independentes e teve a duração de 40 dias. O delineamento experimental adotado foi o inteiramente casualizado (DIC), com cinco tratamentos e quatro repetições, totalizando 20 unidades experimentais. Foram utilizadas 1.400 larvas de acará bandeira (Pterophyllum scalare) divididas entre os tratamentos. Os tratamentos consistiram em: 0, 50, 100, 200 e 400 mg de extrato de alho por quilo de ração. Os resultados mostraram que não houve efeito significativo dos níveis de inclusão do extrato do alho sobre os índices zootécnicos avaliados e posteriormente no transporte dos juvenis. Também não foi observado influência na sobrevivência das larvas durante o período experimental. Novas pesquisas com dosagens maiores de extratos devem ser realizados para melhor elucidação do efeito do extrato de alho como promotor de crescimento. We used 1,400 angelfish larvae (Pterophyllum scalare) divided into five treatments with four repetitions each, for a total of 20 experiments. The treatments consisted of five isoprotein and isocaloric diets: T1= 0 mg/kg, T2= 50 mg, T3= 100 mg/kg, T4= 200 mg/kg, T5= 400 mg/kg of garlic extract per kilogram of feed. The experimental replicates were randomly distributed in the recirculation system.

Palavras
The manufacture of the feed took place using industrial type procedures involving all the basic processes of manufacturing a commercial feed, including the extrusion process. For the ingestion of the feed by the larvae, and later on in their development as juveniles, the feed was ground and sieved in order to allow us to obtain smaller particles. The correction of feed granulometry occurred with the correction of larvae length.
The composition of the feed used for the present study followed the necessary premises for the basic nutrition of fish, with experimental values of garlic extract included. The composition of ingredients can be observed in Table1. The experimental design adopted was completely randomized (RED), with five treatments, four replicates, the production process (Sales and Janssens, 2003). Stress in fish promotes biochemical and physiological changes, which start with the release of catacolamines into the circulation, followed by the release of cortisol, ionic imbalance, changes in hematological parameters, physical exhaustion, loss of organic function, immunosuppression and death (Aride et al., 2016(Aride et al., , 2018Leal et al., 2020;Wendelaar Bonga, 1997).
Studies have been carried out with plant extracts that have been included in commercial or experimental feed for fish in order to obtain better results in weight gain and immune responses, as well as to combat various diseases caused by a variety of infectious agents, and thus optimize production and avoid losses (Shalaby et al., 2006). Herbal and vegetable additives act as growth promoters and their use has been observed since ancient times. Over time, our knowledge regarding plants has evolved, and systematic isolation and characterization of the active ingredients contained in these plant sources has been achieved (Costa et al., 2007). One of these natural additives for use in growth promotion is garlic extract.
Garlic (Allium sativum) is a plant that originates from Asia and has been used in the therapy of many diseases since ancient times and its cultivation and drug use is known worldwide (Sobrinho and Lopes, 1993). According to Kemper (2008), its uses range from being an appetite stimulant to an antibiotic. Several studies have been carried out with the use of garlic in animal treatment, highlighting use directly in water to control the ectoparasite Trichodina spp in juvenile tilapia (Chitmanat et al., 2005), for parasitic control in sheep, (Sunada et al., 2011), and for the control of bacteria in tilapia (Oreochromis niloticus) (Hussein et al., 2013).
These characteristics make garlic extract a promising additive to be more thoroughly studied in fish nutrition, with the intention of seeking both to minimize losses due to mortality during cultivation and transport, as well as to improve the zootechnical performance of these animals. The objective of this study was to evaluate the properties of garlic as a promoter of growth and survival in angelfish (Pterophyllum scalare) larvae and its benefits in the transport of juveniles of this species.

Materials and Methods
The experiment was carried out in the aquaculture sector of the Animal Husbandry Research Support Unit of the Center for Agricultural Sciences and Technologies, of the North Fluminense State University Darcy Ribeiro, located in the State Agricultural Technical School Antonio Sarlo, Campos dos Goytacazes, Rio de Janeiro.
The fish used were obtained from four natural spawns of the same age from matrices belonging to the aquaculture sector's collection. The experiment was conducted in a recirculation system composed of 20 aquariums of 40 L each, and were equipped with independent water inlets and outlets. The system was also composed of a mechanical and biological filter. To maintain the temperature within optimal ranges for the species two thermostats were used. totaling 20 experimental units where the variables that were measured directly in the animals were analyzed according to the methodology of mixed models based on the following statistical model: Where ijk Y corresponds to the measurement performed in the k-th larva, inside the j-th aquarium that received the i-th treatment, and represents the random effect of the j-th aquarium within the i-th treatment, this supposed effect is normally distributed with a mean of 0 and variance 2 a σ . The term ( ) k ij e represents the normal and independently distributed supposed random error, with mean of 0 and variance 2 σ . The effect of treatments was tested using the estimate of the variance of 2 a σ as the denominator. The assumption of homoscedasticity for 2 a σ was verified using a simple model containing only one variance for the different treatments and a model containing heterogeneous variances for the different treatments. The Akaike criterion (1974) was then calculated, and corrected for finite samples or AICc (Sugiura, 1978), according to the recommendations of Burnham and Anderson (2004), and took as the best model the one that presented the highest chance of likelihood and the criterion of parsimony for the degree of parameterization of the models. Variables involving concentrations, i.e. specific growth rate and survival, were transformed to fit the normality criterion. The transformed variable, i.e.,

sin
performed. In both cases, the MIXED procedure of the SAS statistical program was used (Version 9, SAS System Inc., Cary, NC, USA). The larvae were weighed and measured to obtain the initial weight (IW), height (H), total length (TL) and standard length (SL), with the aid of a precision analytical scale and caliper. After the measurements were divided between the treatments so that the number of fish from each spawning was equal among all repetitions, thus decreasing the effect of genetics on the final result. The total was 70 larvae per replicate. The animals, after biometrics performed at the beginning of the experiment, presented mean values for IW= 9.7±0.20 mg; H= 3.71±0.70 mm; TL= 9.86±0.64 mm; SL= 7.84±0.65 mm.
The monitoring of the physico-chemical variables of the water occurred once every day during the length of the experiment, with the temperature, pH and dissolved oxygen regulated to values within acceptable limits for the species studied.
The feeding of the diets occurred three times a day, ad libtum, at 8:00h, 12:00h and 17:00h. One hour after each meal, the aquariums were siphoned for the withdrawal of the leftover feed to avoid the depreciation of the quality of the physico-chemical variables of the water and to avoid a possible leaching of the garlic extract.
To evaluate the effects of garlic extract in transport, at the end of the experimental period (40 days), six juveniles were taken from each replicate and were stored in packs with 500 ml of water, according to the ratio of two thirds of air to one of water. In the packaging, pure oxygen was not added. The oxygen dissolved in the water in the packages at the time of closing was measured and presented a mean value of 6.8 mg/L, which was considered sufficient for the survival of the juveniles for a satisfactory time. This permitted us to evaluate the effect of the garlic extract diets on transport.
The start time of the experiment was determined as being when the packaging was sealed, and the end was considered when the last animal died. The time of death of all specimens was determined for subsequent statistical analysis. The packages were examined every hour until the last specimen died, in order to establish the total time of possible transport.
After a normality test had been performed, the treatment groups were compared using one-way ANOVA and the Tukey test was used for post-hoc comparison of means at P < 0.05.

Results
During the experimental period, the water temperature was 27.3 ± 1.13 °C, which is considered within the comfort range for the species. The mean dissolved oxygen level was 3.91 ± 0.91 mg/L, the mean pH value was 7.22 ± 0.87, which are values considered satisfactory for the species.
Among the treatments evaluated, the one that contained levels of garlic of 200 mg/kg of feed obtained better performance for final weight (Figure 1), total length (Figure 2), standard length ( Figure 3) and height (Figure 4), when compared to the other treatments, however differences were not significant (p>0.05). The survival found ( Figure 5) as a result of the treatments was higher in the treatment with inclusion of 400 mg/kg of feed, followed by the control treatment in which there was no inclusion of garlic extract, and the difference among the treatments was not significant (Figure 6) (p>0.05). This result suggests that no interference was caused by the different levels of garlic extract, since the highest values of zoo technical performance were observed with the highest and lowest level of inclusion of garlic extract respectively.
When we submitted the data to statistical analysis, it was observed that there was no statistically significant difference among the treatments (p>0.05) for any of the parameters of zoo technical performance.

Discussion
Similar results were observed with juvenile tilapia hybrids (Oreochromisniloticus x O. aureous), since Ndong and Fall (2007) evaluated the inclusion of levels of 0, 0.5, and 1% of raw garlic in experimental feed and also did not observe significant effect of garlic-rich diets on animal performance. However, the same author evaluated the response of the immune system in relation to the different levels of inclusion of garlic and observed a significant effect in the amount of leukocyte, phagocytic activity, and lysosomal activity of animals fed 0.5% of garlic in the feed.     response to stress conditions arising from transport, due to a possibly improved immune response, in view of the immunostimulating properties of allicin, as suggested by Butolo (2010).
It is possible to conclude that the levels of 0, 50, 100, 200 and 400 mg/kg of garlic extract included in extruded feed were not satisfactory for the improvement of the zootechnical parameters of angelfish larvae. Regarding transport, the inclusion of garlic extract showed no benefits, and the use of it in animal feed in the pre-transport period is thus not recommended. Thus, additional research is needed with other ingredients that promote benefits in the pre-transport of angelfish larvae. CHITMANAT, C., TONGDONMUAN, K. and NUNSONG, W., 2005 The non-observation of significant difference among treatments may have been influenced by the absence of pathogenic organisms and stressors throughout the experimental period, considering that some of the actions of allicin in the animal's organism is the action against pathogens and immunostimulants (Butolo, 2010). As the cultivation system had strict control of the physico-chemical variables of the water throughout the experimental period, which kept them within comfortable limits for the studied species, it may have contributed to a possible absence of a significant effect of garlic extract on the performance and survival of the larvae. Marchiori (2005) concluded that there is an immediate need for consumption immediately after preparation with regard to garlic and its beneficial properties, and without the action of heat or any other type of heat treatment, which greatly decreases the concentrations of the sulfide phytochemicals in question. Since the experimental feed was processed and extruded with the presence of heat in the process, substances that could be beneficial to fish performance may have been inactivated or have had reduced action, resulting in non-significant differences among treatments.
Although no positive values for performance were observed in this study, other authors have demonstrated positive results for the use of garlic in fish performance. Shalaby et al. (2006) observed a positive result in the performance of juvenile tilapia, where levels of 1, 2, 3 and 4% of garlic extract were included in an experimental feed. It was observed that the treatment in which 3% of garlic extract was included had better results than the control treatment, both in relation to performance and survival.
Studies that showed a positive effect of the inclusion of garlic in the diet were performed with inclusion values that were higher than those tested in the present study. Low inclusion values may have been one of the factors that contributed to the non-significant result among treatments for zootechnical performance, thus requiring more studies focused on the dosage of the garlic extract.
The total transport time values were 174.58 h for the inclusion level of 0 mg, 163.25 h for 50 mg, 134.71 h for 100 mg, 143.17 h for 200 mg and 140.57 h for 400 mg. After the data were submitted to statistical analysis, statistical significance was observed among the treatments evaluated. The treatments where the diet contained a lower level of inclusion presented greater total transport time, as can be seen in the chart below.
This result may have been influenced by the bio stimulator characteristic of garlic extract (Kemper, 2008), in which it is recommended that the rate of oxygen use is higher at higher levels of inclusion. This becomes negative for good practices of fish transport, where one of the priorities is the reduction of the metabolic frequency of the fish. This can be either by using anesthetics or decreasing the temperature, for example, and allow a slow reduction of dissolved oxygen and reduction in the volume of excrement in water, which in a confined environment can become lethal to fish (Gomes et al., 2001;Carneiro and Urbinati, 2001;Inoue et al., 2002). However, the results obtained contradict the hypothesis that fish fed with higher levels of garlic extract could present a better