BA inclusion in the commercial diets is a simple process where original components are not altered. The shrimp-maintained interest in the feed during the 30 days of experimentation were always active at feeding time, similar to what was reported in feeding bioassays when citric acid and malic acid were included in tilapia feed (Adams et al., 1988) and with several other organic acids (OAs) such as sodium (Na) salts for P. vannamei (Silva et al., 2013). In addition, greater palability was obtained by including these OA in the feed.
The BA under the evaluated conditions produced no change in water quality with a total refill of 9.5 times day− 1 during the trial period. The physio-chemical parameters during the 30 day trial were maintained with optimal values for the growth of juvenile white shrimp (P. vannamei) with salinity 33 ± 2 UPS, temperature 30 ± 1°C, pH 7–9, OD 5 ± 1 mgL− 1, nitrites < 0.1 mgL− 1, nitrates 0.4–0.8 mg L− 1, ammonium 0.1–1 mg L− 1, turbidity 35 to 45 cm, and alkalinity 100–140 mgL− 1 (Carbajal et al., 2013; Lim and Dominy, 1990, https://www.gob.mx/inapesca/acciones-y-programas/acuacultura-camaron-blanco-del-pacifico).
Our results show that survival was not affected between the different concentrations with 98.89% in C0, C2, and C3 and 94.44% in C1, the lowest value in C1 can be attributed to management and not to the concentration of BA; similar results were reported in Anuta et al. (2011) when evaluating acidic calcium sulphate. However, Churchird et al. (2015) obtained higher survival values by including formic acid in the post larvae diet of P. vannamei, even after being exposed to Vibrio parahaemolyticus. In addition, similar results mention Su et al. (2014) using citric acid. The above studies highlight the importance of what is mentioned by Ng et al. (2017), who recommend that OA should be tested on different species and development stages because the effects varied a lot.
It is important to emphasize that, after the stress test, all concentrations had 100% survival which suggests that osmotic adaptability and health status of the shrimp was not affected due BA presence in the feed. However, the evaluation of adequate concentrations of BA in P. vannamei is crucial. In the present study, it was shown that the inclusion of BA in the feed favors the development of alterations in shrimp antennal gland atrophy and development of lesions in hepatopancreatic tissues as in agreeance with Puello-Cruz et al. (unpublished observations) with higher concentrations. In addition, this study showed that as the concentration of BA increased, growth decreased. A similar result was reported by Yao et al. (2019) by using micro-encapsulated salts of OA in combination with enzymes. Contrary, da Silva et al. (2014) obtained better growths when using butyrate and propionate in P. vannamei.
OA and their salts have demonstrated the ability to improve digestibility and gut health in terrestrial and aquatic species (Ng et al., 2017). FAO (2009) reports feed conversion rates (FCR) for P. vannamei from 1.2 to 1.8. Our BA concentrations remained within reported levels (C1: 1.9, C2: 1.,8 and C3: 1.9), however, the control (C0: 2.4) was significantly higher (Table 1). This suggests that BA promotes the digestive process as it is a precursor to a wide range of primary and secondary metabolites. BA is found naturally in plant and animal tissues, and plays important roles in growth (Zuo et al., 2002; Qualley et al., 2012; Olmo et al., 2015). Silva et al. (2015, 2016) and Romano et al. (2015) reported improvements using Na-propionate and OA mixtures also with P. vannamei. Ng et al. (2017) reported that OA improved the availability of nutrients in the feed by pH reduction, causing greater absorption, and decreasing bacterial communities that could compete for the absorption of nutrients in aquatic animals. Therefore, due to all of the controversial results from other studies, it was recommended to carry out specific digestibility and metagenomics studies to evaluate BA effects in shrimp.
On the other hand, there is evidence that the effects on organisms vary according to the OA used, species, and developmental stage (Cuvin-Aralar et al., 2011; Ng et al., 2017). We suggested feeding frequency and duration periods to also be evaluated in further research as well. To date, most studies of BA included in diets have been done in terrestrial animals. Giannenas et al. (2014) and Kiarie et al. (2018) obtained growth benefits in pigs and turkeys promoting intestinal health. However, Bühler et al. (2006) reported a negative effect on weight gain and feed conversion in piglets, comparable to those determined in this study regarding the shrimp growth (weight and length) which, at higher BA concentrations included in the feed, reported lower growth (Table 1).
Energy content and BA concentration in muscle results did not show a significant variation before or after feeding with the different concentrations, suggesting that non-conserved energy benefits and/or were not the correct amount for tissue accumulation and/or the organism could discard them. In broiler chickens, it was shown that concentrations greater than 0.1% of BA in the feed reduced growth rate and were discarded in the urine. This suggests a possible route of elimination by shrimp (Nesheim et al., 1963). Regarding the ability of several OA to easily dissociate and leach completely within 30 minutes of the pellets in water contact, Ng et al. (2017) and da Silva et al. (2013) revealed that it may also be either or both of these two reasons that BA has not been detected in shrimp muscle at any concentration used in this study. Even though these BA concentrations report damage in hepatopancreas analysis, for accurate results, they have to be included for further evaluations.
In decapod crustaceans, the antennal gland plays a very important role in nitrogenous compound excretions and, together with the gills and intestines, participates in osmoregulation (Tsai and Lin, 2014). In this study, histopathological analysis showed tissue alterations such as antennal gland atrophy when BA was included in feed, contrary to what was observed in shrimp fed without BA. These results show a direct effect on the antennal gland and confirm a chronic effect, possibly due to the constant demand to remove BA. It will be interesting to carry out studies focused on understanding the mechanisms that influence cell damage and evaluate the excretion mechanisms of nitrogenous compounds and their effect on osmoregulation.
Hepatopancreas negative effects were higher as BA concentrations increased, such as lipid reduction, and histological damage (atrophy, hemocytic infiltration, and necrosis) (Figs. 2 and 3), contrary to what was reported by Romano et al. (2015), which reported high lipid concentrations in hepatopancreas when using microencapsulated OA mixtures with P. vannamei even after being infected with Vibrio harveyi. The composition and metabolism of lipids in wild crustaceans compared to those subjected to culture can contribute to a better understanding of the influence of nutritional factors on metabolic capacity, which allows for the evaluation of feed characteristics and other environmental factors in farmed animals (Stroud et al., 1982). Generally, lipid level in tissues and organs can vary depending on diets, environment factors, and physiological status and developmental stage (Oliva et al., 1989). In this study, the variation in BA concentration in the diet resulted in lipids depletion in the Hp as the concentration of BA increased. According to our observations, there is a direct relationship between the BA inclusion and shrimp damage (Figs. 1 and 2). The average values of Hi show value increases, contrary to what was observed in treatment without BA (Fig. 3). Lipids are required as energy for shrimp, they are also used for physiological processes such as osmoregulation, enzymatic activity, transport of ions, fatty acids, water permeability in the gills, and in the synthesis of amino acids (Faleiros et al., 2018). In shrimp, it has been shown that under fasting and stress conditions by manipulation for a 3–4 weeks period, lipids are degraded to generate energy, decreasing lipid reserves (Mercier et al., 2006). Our results suggest that this BA concentrations in feed can cause stress and sub-lethal effects in shrimp, as was observed in previous studies with chickens (Amaechi and Anueyiagu, 2012) where they observed mortality, significant growth reduction, and effect on the size of the organs when they were fed with inclusion levels greater than 1.2% of BA and conclude that this was due to toxicity. In our study, although a mortality associated with the concentration of BA was not identified, when performing analyses directly in the Hp, negative effects on the health status of the organisms were observed by increasing the concentrations of BA. A significant decrease (p > 0.05) in the HIS% values and lipid content was recorded. Studies will be needed to understand the mechanisms involved in these effects. Wang et al., 2009 reported that excess free radicals can cause tissue damage, therefore furthering the need for BA research.
In the intestine, although the development of hemocytic enteritis occurred only in shrimp fed with BA, it was not related to concentration. In shrimp, the development of hemocytic enteritis has been associated with poisoning by cyanobacteria (Lightner, 1996) or heavy metals (Frias-Espericueta et al., 2008), suggesting that BA may be exerting an irritating effect on the intestinal epithelium, possibly due to its ability to lower pH in components of the digestive tracts (Amaechi and Anueyiagu, 2012; Diao et al., 2014). There is evidence that prolonged exposure to gradual pH changes (low or high) increases mortality, reduces weight gain, and causes histopathological damage to the Hp and intestine of L. vannamei (Han et al., 2018). Li et al. (2019) observed that the Na+/H + exchanger proteins present intestinal shrimp cell participates in internal cellular re-alkalization and play an important role in the pH regulation of P. vannamei hemolymph when exposed to an acidified environment. Wang et al. (2009) observed that P. vannamei Hp cells and hemocytes exposed to 5.6 and 9.3 pH developed DNA cell damage, while the control treatment (7.4 pH) showed no effect throughout their study. It is possible that, derived from a constant acidification of the digestive tract by the intake of BA shrimp physiology compromised, particularly in the metabolism ions, could explain the histopathological damage in the intestine and antenal gland.