INTRODUCTION

Global fish production has increased steadily over the past five decades and the supply of edible fish has increased at an average annual rate of 3.2%, thus outpacing the global population growth rate of 1.6%. Per capita consumption internationally increased from an average of 9.9 kg in 1960 to 19.2 kg in 2012 (^{FAO, 2018}). Specifically in Mexico, during the last decade the consumption of aquaculture and fishery species has been increasing. Currently, the main aquaculture species in the country are shrimp (150 thousand 76 tons), mojarra tilapia (149 thousand 54 tons), oyster (45 thousand 148 tons), carp (30 thousand 300 tons) and trout (7 thousand tons) (^{CONAPESCA, 2018}).

Regarding the production and market of bullfrog (Lithobates catesbeianus), statistics are scarce. Even so, the Food and Agriculture Organization of the United Nations (^{FAO, 2018}) reports that in the year 1980, it was estimated that 3% of the global frog market (all species) was supplied by aquaculture; while the contribution for the year 2002 was estimated at 15% (taking into account the calculated growth rate of the industry). Taiwan, Brazil and Mexico are the main producers of live frogs (capture and aquaculture). Some documented statistics place the United States of America as the largest consumer of frogs, followed by France and Canada; with three main market niches: frog legs, live frogs and frogs for educational and scientific needs (^{FAO, 2009})

In Mexico, bullfrog (Lithobates catesbeianus) production is led by the Mexico State of, followed by Sinaloa, Nayarit and Jalisco with 35 animal production units, with an average of 60 hectares of surface area used (^{INAPESCA, 2018}). The main aquaculture production systems used in the country are extensive (farming in reservoirs with minimal human intervention after planting and low yields), semi-intensive (farming in ponds, pens and bodies of water) and intensive (farming in controlled systems, ponds, cages, fast-flow channels or water recirculation and reconditioning systems) (^{INAPESCA, 2018}).

While, the demand for live frogs for food has increased, it is expected that conducting research on nutrition, pathology and reproduction will lead to significant improvements that will boost their production. As well as an increase in market prices, because as the trade and capture of wild frogs is restricted, their cultivation increases; however, improvements must be made in marketing, since frog meat and its qualities are far from being widely known (^{FAO, 2009}).

To face these challenges, innovative technological options are emerging that improve production efficiency, in favor of environmental, economic and social sustainability. For although we know that freshwater is a fundamental requirement for aquaculture, it is necessary to recognize that reserves are finite in arid regions and water is scarce, generating competition between productive sectors for the distribution of water resources (^{Neto & Ostrensky, 2015}). Thus, the use of non-conventional water resources in aquaculture is identified as a potential mechanism to improve food production yields while preserving non-renewable and renewable freshwater resources (^{Corner et al., 2020}).

In response to this problem, Biofloc technology emerges as an alternative for water reuse at industrial levels, with a positive impact on the environment (^{Mancipe et al., 2019}) as its application can be carried out in integrated production systems (^{Bossier & Ekasari, 2017}). In the production of aquaculture alternative species, the implementation of a Biofloc system means a reduction of more than 50% of the water footprint involved in production; in addition to creating a positive effect on animal health (^{Bossier & Ekasari, 2017}).

The implementation of such technology in the aquaculture area is based on the creation of a microbiome that reuses organic fish waste and unused feed; creating flocs of bacterial aggregates large enough to be detected by fish and feed them; these microbiota aggregates usually contain protein percentages of up to 27.5% and 7.5% lipids (^{Ekasari et al., 2014}). Being so, these protein and energy levels can even be compared to the quality of commercial feed for production fish.

Considering that microorganisms are an essential part of aquifer ecosystems, their role in nutrient recycling is essential in the trophic chain of systems. For decades they have been used as prebiotics and immunostimulants, for disease control, as well as water quality improvers in aquaculture production ponds (^{Martínez et al., 2017}). Microbial-based systems represent one of the most viable strategies to achieve sustainable aquaculture, as these systems are based on the promotion of microbial proliferation; expecting these to use, recycle and transform excess nutrients from feces, dead organisms, uneaten food and various metabolites into biomass; in addition to displacing pathogenic organisms in production systems (^{Martínez et al., 2015}; ^{Huerta et al., 2019}).

Besides taking into account that Biofloc system application in aquaculture production achieves a nitrification process, this happens through the carbohydrate source that is added to ponds, since it allows bacteria and microorganisms to convert organic waste from feces and wasted feed; decreasing the amount of ammonium, improving water quality and allowing it to be practically eternal in ponds (^{Wei et al., 2016}).

Therefore, the aim of this study was to measure the effect of using reuse water from a Biofloc system for tilapia (Oreochromis niloticus) culture in the intensive production of bullfrogs (Lithobates catesbeianus), as an alternative use for arid and semi-arid zones from Mexico.

MATERIAL AND METHODS

Study area: the study was conducted at the Bullfrog Reproduction, Research and Technology Transfer Center "El Chaveño" in agreement to the Autonomous University of Aguascalientes; located in Jesús María, Aguascalientes, Mexico; with an average annual temperature of 17 ºC, average annual rainfall of 531 mm and located at 1,880 m a.s.l. (^{INEGI, 2021}).

Biological material: a total of 4,000 bullfrogs (Lithobates catesbeiana), with an average initial weight of 49.8 grams/organism, distributed in 40 pens with a semi-flooded system with an effective volume of 400 L, were used.

Experimental design: the experiment was established under a completely randomized design of 4 treatments with 10 replications, obtaining a total of 40 experimental units. Each experimental unit consisted of 100 bullfrogs (Lithobates catesbeiana).

Treatments evaluated: the treatments evaluated were as follows: T1: culture system with weekly 100% fresh water replacement and bottom cleaning. T2: culture system with 30% reuse water from a tilapia (Oreochromis niloticus) Biofloc system and 70% potable water, without water replacement and with the addition of unrefined sugar as a carbon source in a C:N ratio of 15:1. T3: culture system with 60% reuse water from a tilapia (Oreochromis niloticus) Biofloc system and 40% potable water, without water exchange and with the addition of unrefined sugar as a carbon source at a C:N ratio of 15:1. T4: culture system with 90% reuse water from a tilapia (Oreochromis niloticus) Biofloc system and 10% potable water, without water replacement and with the addition of unrefined sugar as a carbon source at a C:N ratio of 15:1.

Production system: the production system used in the study was of the semi-flooded type with uniform confinement surfaces of 8 m², the floodable capacity of each pond was 0.4 m³, with a dry area of 0.4 m² with feeding in the dry area of the floor. With light lamps distributed in the aquaculture production unit to maintain a photoperiod of 14 hours of light (10 hours of darkness), with a temperature between 28 - 42 °C in a 24 hours cycle and a constant water temperature between 26-28 °C. Ambient humidity was maintained at 95- 98% using water sprinklers. The study period was 15 weeks, August - November 2020.

Diets and feeding: an isoproteic and isocaloric diet was used (^{Rincón et al., 2012}), based on commercial feed for trout (Salmo trutta) and catfish (Ictalurus punctatus) Nutripec Purina^® brand with 40% crude protein and 9% fat for the development stage. The amount of ration was supplied once a day (^{SENASICA, 2016}) and it was calculated based on the biomass at a feeding rate of 6% maintained during the experimental period and adjusted 20 days after the experiment starting to 3% of the biomass. For the determination of weight gain, the total weight of bullfrogs was recorded at the beginning of the experiment and weekly with a digital scale with a sensitivity of 0.1g (303D, DESEGO, Mexico).

Zootechnical parameters evaluated: the variables evaluated were: Weight Gain (WG) with the formula WG= FWIW, where FW is Final Weight and IW is Initial Weight. The Specific Growth Rate (SGR) was calculated with the formula SGR (%) = (Ln) (Fw)-Ln (Iw)/tx100; where: Fw and Iw are Final weight and Initial weight, t is time1 and Ln is the natural logarithm of weights. The percentage of Survival (%S) at the period end was calculated with the formula %S= final organism No/initial organism No x 100. Survival Rate (SR) and Feed Conversion (FC) obtained from the ratio between consumed feed and biomass at the end of the experimental period (^{Gutiérrez et al., 2016}).

Water quality: during the study, water quality remained within parameters established for bullfrogs (Lithobates catesbeiana) in intensive production (SENASICA, 2016). The physicochemical parameters evaluated weekly were: temperature (T °C), conductivity (μs), pH and ammonium (mg/l); being taken with multiprobe (556 MPS, YSI, USA). While hardness (mg/l CaCO3) and alkalinity (mg/l CaCO3) were taken with a test kit (FF-1A, HACH, Germany), as described by ^{Plazas & Paz, (2019)}.

Bacterial flocs: for the establishment of bacterial flocs in tilapia (Oreochromis niloticus) culture, it was inoculated with leachate from Californian red worm (Eisenia foetida) litter; for which 3 L of leachate was used for every 10m³ of water in tanks 1L/10m³ of nitrifying bacteria for fish of the PondPerfect 4in brand. For Biofloc establishment, unrefined sugar was used at a rate of 0.02 g/L to ensure a C source and 5 mg/L ammonium chloride (NH4Cl) as an N source; in addition to 2 g/L sea salt and 50 g/L sodium bicarbonate (NaHCO3) to ensure an initial source of alkalinity for the bacteria according to the methodology of ^{Luo et al., (2014)}. Unrefined sugar continued to be added every two days according to the Biofloc volume measured in the Imhoff cones.

Statistical analysis: IBM SPSS Statistic version 27.0.0 was used. In each experience the hypothesis of "the reuse of Biofloc water affects the productive parameters of bullfrog (Lithobates catesbeiana)" was assessed by means of variance analysis (ANOVA) with a confidence level of 95% (^{Ducoing, 2019}), applying a Mauchly's test of sphericity ^{Mauchly, (1940)}. When this null hypothesis was rejected, a Greenhouse-Geisser or Huynh-Feldt test of adjustment was used (^{Bardera, 2019}). When significant overall effects were found, simple effects tests were performed followed by post hoc tests. Post hoc analyses were performed using Tukey's test to analyze differences between treatments with different percentage of reused water from a Biofloc system and drinking water, using Bonferroni pairwise comparisons to test for differences between the behaviors analyzed (^{Bardera, 2019}).

RESULTS AND DISCUSSION

The mean of the average water quality parameters recorded during the study were: temperature 20°C, conductivity 0.4μs, pH 7.2, ammonium 1.19 mg/l, hardness 46 mg/l CaCO₃ and alkalinity 40 mg/l CaCO₃.

Regarding the study variables, results show statistical difference of means between treatments; however, Mauchly's test of sphericity is rejected as it does not show significance. Due to the violation of sphericity, the Greenhouse-Geisser or Huynh-Feldt corrections were applied; which shows significance among treatments in the variables biomass weight, average weight and feed conversion. In turn, in the post hoc test, the difference between treatments under the Bonferroni test showed significant differences for the use of Biofloc 30, 60, and 90% in the variables biomass weight, feed intake and feed conversion, respectively.

Weight gain (WG) behavior (Table 1) showed to be statistically better in the wastewater- based treatments of a Biofloc system; however, this may be related to a higher mortality observed in T1 (drinking water). The estimation of the average weight of marginal means shows no significant differences, suggesting that the average weight of the bullfrog (Lithobates catesbeiana) is not altered with the use of different percentages of Biofloc in the water, as growth rates and weight gain are estimated to be similar in treatments.

The effect on feed consumption (Table 2) is significantly higher in the Biofloc treatments, compared to organisms that received only drinking water; these results correlate with those obtained on feed conversion (Table 3), where a better effect is observed in the Biofloc wastewater-based treatments. The estimates of marginal means for feed consumption show significant differences in T3 (60% Biofloc) and T4 (90% Biofloc), showing a relationship with the higher weight gain observed.

Regarding feed conversion, it is observed (Table 3 and Figure 1) that the organisms of T1 (drinking water) show lower feed conversion than those of T4 (90% Biofloc) during the first weeks of the study; however, after week 8 the conversions are equalized, ending without significant differences by week 15 of the study. It is important to highlight that from week 4 onwards, feed consumption begins to be higher in T2 (30% Biofloc), T3 (60% Biofloc) and T4 (90% Biofloc); this is the effect of the lower mortality and the greater number of surviving individuals and better assimilation of nutrients.

Figure 1 Feed conversion performance by treatment 

Several studies have demonstrated more efficient diet and nutrient assimilation in systems where Biofloc is used (Figure. 1). ^{Da Silva et al. (2013)}, found that the application of Biofloc technology in the intensive culture of Pacific white shrimp (Litopenaeus vannamei) significantly improves the improved efficiency of N and P utilization by up to 70 and 66%, respectively, in relation to conventional intensive culture systems with regular water exchange. Authors such as ^{Mercante et al., (2014)} have described that high levels of phosphorus and nitrogen in the water of intensive bullfrog (Lithobates catesbeiana) production ponds decrease water quality parameters and interfere with productivity; these same effects have been found in the use of Biofloc in tilapia (Oreochromis niloticus) culture (^{Schveitzer et al., 2013}; ^{Widanarni et al., 2012}).

While Table 4 shows a significant difference between means in the specific growth rate in T2 (30% Biofloc) with respect to T1 (drinking water); in the same way this effect is observed in T3 (60% Biofloc). With respect to the effect of treatments on the survival rate (Table 5), the difference between means of the variables T1 (drinking water), compared to organisms of the other treatments, is observed.

The specific growth rate of the organisms of T2 (30% Biofloc), T3 (60% Biofloc) and T4 (90% Biofloc) is higher than in the organisms that received drinking water, which suggests that the microbial diversity in the water has a beneficial effect on the growth and development of this species under intensive production conditions; this effect coincides with the results observed in the survival rate of this study. These results suggest that it is feasible to use water from intensive tilapia (Oreochromis niloticus) farming for reuse in the intensive production of bullfrogs (Lithobates catesbeianus), since the microbial quality existing in the medium benefits interactions with pathogenic microorganisms, decreasing mortality in frogs that receive reuse water in different proportions as happens in other aquaculture species (^{Vinatea et al., 2018}).

It was observed that survival was similar among treatments evaluated, highlighting that in T3 (60% Biofloc) the organisms showed a better survival rate. Results suggest that the great diversity of organisms present in the reused water of a Bioflc system, exert a competition with potential pathogenic microorganisms that attack frogs; this effect has been observed in aquaculture cultures using a Biofloc system (^{Martinez et al., 2016}; ^{Ekasari et al., 2014}). Suggesting that this effect creates a competition of potential pathogenic organisms, reducing their proliferation in the experimental ponds as well as in the digestive tract of the fish (^{Manduca et al., 2021}).

Published studies show that the autochthonous microbiota of the skin and gastrointestinal tract could be affected by many factors, such as microbial interactions, water flows, husbandry, techniques and disinfection; which could alter the balance of microbial ecosystems. These aspects, together with the stress produced by overcrowding, can overcome immune barriers, causing microbial microorganisms to attack, leading to outbreaks of infectious diseases (^{Mauel et al., 2002}); providing bullfrogs (Lithobates catesbeiana) with a microbial environment rich in beneficial microorganisms improves performance in intensive production systems. It has been shown that different strains of Gram (+) as well as Gram (-) lactic acid bacteria isolated from fish cultures have been used for the control of disease-causing bacteria in frogs, such as Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus epidermidis (^{Pasteris et al., 2009}).

On the other hand, ^{Mayorga et al., (2015)}, found that Biofloc was the main food source consumed preferentially by tilapia (Oreochromis niloticus) versus balanced feed. Therefore, it is important to highlight that in Mexico, given the availability of feed (Fattening extruded, at 20 and 25% crude protein El Pedregal and Los Belenes), they can be used in Biofloc culture to minimize the impact of feed cost and take advantage of the preference of tilapia (Oreochromis niloticus), for biofloc; and thus reduce production costs remain preponderant.

There is a scientific reality that indicates the high nutritional content of bioflocs (^{Ekasari & Maryam, 2012}), an aspect that does not seem to apply in Mexico, since most of them use balanced feed with high levels of protein 45/32/25 respectively. When the feed could be eliminated at 32% and used at 25% to favor the consumption of microbial flocs that are preferred by tilapia. Finally, ^{Martínez et al., (2017)}, argue that global evidence supports the hypothesis that the use of microorganisms as a direct feed source in aquaculture will revolutionize the industry, closing the gap towards sustainability.

CONCLUSION

The intensive production of bullfrogs (Lithobates catesbeianus) with reuse water from a Biofloc system for tilapia (Oreochromis niloticus) culture is feasible, since variables evaluated, weight gain, specific growth rate and survival; as well as feed conversion in bullfrogs (Lithobates catesbeianus), showed a positive statistical difference in relation to aquaculture production with potable water reuse, being an option for the efficient use of water resources in arid and semi-arid zones of Mexico.