ELSEVIER Aquaculture 152 (1997) 91 - 101

Use of artificial grazing substrates in bullfrog tadpole culture

Alejandro Flores-Nava * , Eucario Gasca-Leyva Centro de Inuesligaci6n y de Estudios Avanzados del IPN (CINVESTAV), Unidad Me'rida, Apdo. Postal

73-CORDEMEX, 97310 Me'rida, Yucatrin, Me'xico

Accepted 16 December 1996

Abstract

A 45-day experiment was conducted to compare the effects of vertical and horizontal artificial grazing surfaces on Rana catesbeiana tadpole growth and metamorphosis. Groups of Stage 26 tadpoles were stocked at 1 individual I - ' in 10-1 plastic containers. Powdered, moistened feed mixed with gelatine, was adhered to acrylic plates as feeding substrates in TI (vertical, parallel to the side walls) and T, (horizontal, at mid-depth). As a control (C), the same feed was spread homogeneously on the bottom of a third set of containers. Four replicates were used for each treatment. Growth of tadpoles in T, was significantly higher ( P < 0.05) than that in TI or C, with an individual mean weight at metamorphic climax of 3.2 g (f 0.04 s.e.1, as opposed to 2.3 g ( + 0.05 s.e.1 and 2.1 g ( + 0.07 s.e.), respectively. The percentage of metamorphosed individuals was also significantly higher ( P < 0.05) in T, (81%), than in T , (73%) and C (55%). Respective food conversion ratios for T,, T, and C were 2.40, 2.82 and 2.65. O 1997 Elsevier Science B.V.

Keywords: Tadpole; Bullfrog; Feeding; Substrate

1. Introduction

The feeding of tadpoles in bullfrog farming operations is of crucial importance, not only in terms of quantity and quality of the food fed, but also in terms of its physical form or presentation. The most common presentations currently employed by commer- cial farmers are powdered feed (Lopes-Lima and Agostinho, 1988, 1992), moist doughs (Flores-Nava, 1994; Flores-Nava et a]., 1992, 1994) and pellets of less than 32 mm in diameter (Fontanello et al., 1985).

Corresponding author

0044-8486/97/$17.00 O 1997 Elsevier Science B.V. All rights reserved. PI1 SOO44-8486(97)00005-7

Several experiments have compared growth and metamorphosis of Ram catesbeiana tadpoles fed different presentations, and the results indicate that moist doughs are most effective as compared with powdered or pelleted feeds (Mazzoni et al., 1992; Agostinho et al., 1995).

However, there are other practical problems associated with feed presentation in tadpole rearing, which in the end influence the overall performance of the cultured organisms. These include deterioration of water quality due to uneaten food particles (Flores-Nava, 1992), increased labor for cleaning the tadpole tanks and, most impor- tantly in our own experience, localized concentration of food in the tanks, leading to increased competition.

Considering the ability of ranid tadpoles to scrape solid surfaces (Dickman, 1968), artificial, removable grazing substrates appear to be an alternative to conventional food presentation, thus helping to improve both management of rearing tanks and use of feed by tadpoles.

Artificial grazing substrates have been used mainly in gastropod farming where feeder plates provide an adequate amount of feed in relation to the density of the cultured organisms (Fallu, 1991), and this method has also been tested in tilapia production (Shrestha and Knud-Hansen, 1994). This technique can be applied to other species, including bullfrog tadpoles.

The present work was carried out in order to assess the effects of artificial grazing substrates on the culture of bullfrog, Rana catesbeiana, tadpoles.

2. Materials and methods

2.1. Experimental procedure

A total of 120, Stage 26 (Gosner, 1960) (0.33 + 1.015 mg s.e. per individual) R. catesbeiana tadpoles obtained from an egg mass (same cohort) collected in Morelos State, Central Mexico, was used. All references to tadpole stages in this paper conform to the Gosner (1960) criteria, which consider 46 stages based on morphological characteristics from fertilization (Stage I), through to Stage 42 (beginning of the metamorphic climax) beyond which total resorption of the tail is completed in Stage 46.

The organisms were stocked at 1 1-' in 10-1 (0.25 m X 0.4 m X 0.10 m) rectangular plastic containers. The experimental design consisted of two treatments and a control (Fig. 1): T,, containers with grazing substrates placed at mid-water (water column depth = 0.10 m) with food on dorsal position; T,, containers with grazing substrates placed vertically, covering the four walls of the container; control (C), containers where the feed (same amount as in T, and T2) was homogeneously distributed, directly on the bottom of the container (conventional feeding method) (Lopes-Lima and Agostinho, 1988; Flores-Nava, 1992). The plates were positioned in the containers so that all tadpoles had the same opportunity to get access to the feed. Four replicate tanks were used for each treatment and the control.

Purina shrimp feed (Purina, S.A., MCxico) was used in the experiment, the proximal analysis of which is presented in Table 1. The feed was ground and sieved through a

ON BOTTOM Fig. 1 . Position of horizontal (T,) and vertical (T?) grazing substrates in the experimental units.

0.250-rnm mesh. Forty grams of this powdered feed were then moistened by adding 90 ml of water. Gelatine (Knox brand, Naucalpan, Mexico) was added as a binding and adherence agent at 4% of the total amount of feed (dry basis included in the proximal analysis). The feed was then weighed out according to tadpole biomass (10% body weight day-') and allowed to solidify in a refrigerator for 30 min at 10°C. Finally it was spread onto rectangular 22.8 X 4.7-cm acrylic plates.

Every day at noon, all the acrylic plates were removed and new plates with the corresponding amount of feed were substituted. The remains of the feed on the plates and on the bottom of the containers were carefully syphoned, dried at 95 5°C for 24 h and re-weighed in order to calculate the actual food intake.

Experimental containers were kept indoors in the frog culture facility of CINVES- TAV-Mtrida. All containers were provided with light aeration and were subjected to natural photoperiod (12 L:12 D). Tap water (initial water quality: 220 mg CaCO, 1-' total hardness; 25S°C; 0.013 mg NH,-NH,-N I-'; 6 mg dissolved oxygen 1-'1 was used in the experiment. Water in the containers was exchanged (50% every day) at noon by careful syphoning.

Table 1 Proximate analysis composition of one sample of the Purina shrimp feed added with gelatine

Percentage (wet basis)

Moisture Lipids Fiber Protein Ash Nitrogen-free extract

Water quality parameters were monitored during the experiment: dissolved oxygen (DO); temperature and pH (registered daily by means of a Beckman Model 32 Multimeter). Nitrite and total ammonia (NH, + NH,) were determined weekly in all

r containers, using a Shimadzu 1201 spectrophotometer, reading samples at 543 nm, after the formation of the diazo compound, with sulfanilamide-N-naftil in the case of nitrite, while samples for total ammonia-N were read at 640 nm after the formation of indophenol blue with hypochloride and phenol, following procedures modified from American Public Health Association-American Water Works Association, APHA- AWWA (1985). Water samples were taken by careful syphoning, avoiding settled particles.

Every 15 days, all organisms in each container were weighed in a digital electronic balance to the nearest 0.1 g, after thorough drying in absorbent paper towels. The mean weight per container was calculated, and the amount of feed to be provided was adjusted. The experiment lasted for 45 days.

2.2. Statistical methods

The results were analyzed in terms of body growth, number of animals that metamorphosed (reached Stage 42, with forelimbs and tail) and feeding efficiency.

Normality of the data was evaluated by using Rankit plots (RP) (Sokal and Rohlf, 1979). Bartlett's test was applied for evaluating variance homogeneity. The F,,, test was applied to determine significant differences ( P < 0.05) among treatment variances (Sokal and Rohlf, 1979). Percentage values were arc-sin transformed and then compared

growout period days

Fig. 2. Growth of Rana catesbeiana tadpoles provided with horizontal grazing substrates (T, ), vertical grazing substrates (T2) and no grazing plates (C fed directly on the bottom). Error bars represent + s.e.

through a one-way analysis of variance (based on the fact that heteroscedasticity can be neglected when sample sizes are similar) (Norusis, 1988), and means compared for significance (P < 0.05) using Duncan's multiple range test (DMRT; Duncan, 1955).

Control

I n

Treatment 1

7 1 n

Treatment 2

Fig. 3. Size-frequency distributions of tadpoles feeding on horizontal (T,) and vertical (T2) grazing substrates, and directly on the bottom (C). ( n = 40 for each treatment.)

Table 2 Survival, growth and feeding efficiency of Rana catesbeiana tadpoles raised on horizontal (T2) and vertical (TI) artificial grazing surfaces, and fed directly on the bottom (C). n = 4

c TI T2 + s.e.

Survival (%) 100.00 100.00 100.00 -

Initial body weight (g) 0.32 " 0.35 a 0.33 a 0.01 5

I Final body weight (g) 2.30 2.09 3.20 " 0.056 Specific growth rate (% day - I ) 3.28 a 2.98 a 3.78 a 0.145 Feed intake (g day ) 5.25 a 4.91 a 6.90 a 0.262 FCR 2.65 a 2.92 2.40 a 0.064

Values read horizontally with the same superscripts are not significantly different, based on DMRT (P < 0.05).

Mean figures correspond to each set of data derived from each of the experimental units (i.e. tanks). Means are given f s.e.

3. Results

Growth in body weight of the experimental tadpoles is presented in Fig. 2. Tadpoles of T, showed a significantly ( P < 0.05) higher final mean weight of 3.2 (k0.04) g. Tadpoles in the control averaged 2.3 g (k0.07) and 2.1 g (k0.05) in TI , these results not being significantly different ( P > 0.05).

Fig. 3 presents the size-frequency distributions of tadpoles in each of the three treatments. Even though there was an apparent bimodal pattern graphically observed in T, and C, no deviations from normality were statistically found (values of Wilk-Shapiro RP were: TI = 0.959; T, = 0.945 and C = 0.912).

Table 2 presents the results of growth, feed utilization and survival of the tadpoles used in the experiment. Survival was 100% in all containers. Weight increment was significantly higher ( P < 0.05) in T, (969%) than in TI (597%) and C (718%). Feed

Treatments

Fig. 4. Percentage of metamorphosed individuals (Stage 42 = froglets with four limbs and a tail; Gosner, 1960) on Day 45 of the experiment. C, feeding directly on the bottom of the tank; T , , feeding on horizontal grazing substrates; T2, feeding on vertical grazing substrates. Error bars represent +s.e.

Table 3 Minima, maxima and average recorded figures of water quality parameters from the experimental containers with Rana catesbeiana tadpoles

Units Min. Max. Average n + s.e.

Temperature "C 25.0 28.0 26.5 135 1.70 Oxygen mg 1-' 4.4 6.1 5.16 135 0.55

PH 7.06 8.03 135 NH, +NH,-N mg 1-' 1.66 2.09 2.12 27 0.93 N0,N mg 1-' 0.35 2.27 1.28 27 0.90

conversion ratio (FCR) was more efficient in T, (2.40:l.O) than in T, (2.82:l.O) and C (2.65: 1.0), although the values were not significantly different.

Fig. 4 shows the percentage of metamorphosed individuals recorded in each treatment by Day 45 of the experiment, that is, when they reached Stage 42 (metamorphic climax). More than 80% of the individuals of all replicates of T, reached Stage 42 before Day 45 of the experiment, followed by TI (73%) and the control (55%).

Table 3 presents the water quality data recorded during the experimental period. All measurements remained with little variation for each parameter. Dissolved oxygen was always above 4 mg 1-' and temperature fluctuated between 25 and 28°C.

4. Discussion

Tadpoles that had access to vertical grazing substrates (T,) showed much better growth and feeding performance than those feeding on horizontal substrates (T,) or directly on the bottom of the culture container (C). Graphic comparison of the size distributions also shows more homogeneous growth in tadpoles of T, than those of T, and C. There are two likely explanations for these results.

There is recent evidence that one or several growth-inhibiting agents may be present in feces of both wild and cultured anuran larvae, among which the algae Prototheca sp. appears to be one of the most important of all (Beebee, 1995; Griffiths, 1995). Since coprophagy is a common feeding strategy in ranid tadpoles (Steinwascher, 19781, feeding on the bottom or, as in this experiment, in a horizontal plate, normally results in the ingestion of fecal matter. It is thus possible that such fecal matter could include growth-inhibiting agents. This could explain why those tadpoles scraping vertical plates did not have to search for food on surfaces where it normally mixes with feces and hence may not have been subjected to growth inhibitors. This explanation can also be supported by feed intake (FI) and food conversion ratio (FCR) results, which although were not statistically different among treatments, showed that with less food ingested, a higher weight gain was achieved by using vertical substrates. However, further research is needed in order to elucidate the actual role of fecal matter (including the search for Prototheca sp.) in retarding growth under the experimental conditions.

98 A. Flores-Naua, E. Gasca-Leya / Aquaculture 152 (1997) 91-101

A second explanation for the better performance of T, with respect to T, and C, is that tadpoles feeding on vertical surfaces have to spend their energy more efficiently trying to maintain their bodies in a vertical position in order to get enough feed particles. This way displacement of potential competitors commonly seen under conventional feeding conditions is reduced (Vera, 1994), thus growth is homogeneous.

A normal procedure in aquaculture is to provide cultured tadpoles with a predeter- mined amount of feed per day in relation to their body weight (Lopes-Lima and Agostinho, 1988). This strategy assumes that all individuals will get an equal portion of feed to meet their nutritional needs. Nonetheless, in a practical culture situation, this becomes a problem since competition for food is increased by increased density, thus propitiating intraspecific segregation and asynchronic growth (Collins, 1979; Vera, 1994).

P. Vera and A. Flores-Nava (unpublished, 1996) studied the feeding behavior of R. catesbeiana tadpoles under different stocking densities, and found that, regardless of density, after 3-4 weeks in the rearing tanks, larger tadpoles of the same cohort will out-compete smaller individuals, thus displacing them to the comers and walls of the culture tanks. This phenomenon is accentuated by the use of 'feed balls' randomly placed on the bottom of the tanks, thus concentrating the food in certain points to which a limited number of individuals will have access.

During the present experiment, both control tadpoles and those feeding on horizontal plates displayed the same feeding pattern found in the aforementioned study by Vera and Flores-Nava, whereas those organisms feeding on vertical plates were observed to be more homogeneously distributed over the plates, thus all individuals had access to feed particles. This may explain the better feeding efficiency values in T,, reflected in the lower FCR.

There is a direct positive relationship between stocking density and intraspecific competition in bullfrog tadpoles (Justo et a]., 1985; Vera, 1994). Since in the present experiment a density of only 1 tadpole 1-I was used (a standard density for bullfrog tadpole rearing; Flores-Nava, 1997), further research is needed to determine the effi- ciency of artificial grazing substrates under higher density conditions.

Even though the size of tadpoles was normally distributed in all treatments (Wilk- Shapiro RP = 0.912-0.959), Bartlett's test did show heteroscedasticity (x,?,,,,, = 7.53). F,,, tests revealed that the variance of T, (0.08 g2) was significantly lower (F,,, ,,,,

= 2.39) than C (0.20), and although the variances of TI (0.12) and T, were not significantly different, the smaller value does suggest that tadpoles in T, grew more homogeneously, supporting the explanation of competition.

As far as water quality is concerned, all parameters analyzed were within the adequate ranges reported for tadpole culture (Culley, 1991; Flores-Nava, 1997), indica- tive that the 50% water exchange rate was sufficient to maintain adequate water quality conditions.

It is necessary, however, to carry out other experiments in larger, commercial tanks since suspended solids, which are generated from uneaten food, must be considered when comparing the feeding methods tested in the present trial.

In a more practical context, we also wanted to assess whether the use of artificial feeding substrates could reduce the time of feeding and cleaning of the rearing tanks.

A. Flora-Naua, E. Gasca-Leyua /Aquaculfure 152 (1997) 91-101 99

Table 4 Comparison of the estimated time needed (mid to carry out feed preparation, feeding and tank cleaning, using artificial grazing substrates and direct feeding (estimates were made from routine operations at CINVESTAV's tadpole rearing facilities and this experiment)

Task Feeding substrates Moist doughs

Feed preparation 20 Feeding 10 Cleaning (syphoning) 12 Total 42

Table 4 shows the results of time needed to carry out such tasks. The comparison was made between T, and C, based on approximate averages. Although the dimensions of the experimental units are not representative of those of commercial units, and many other factors such as nature of feed and suction power of the syphon may influence the results, the figures do provide an idea of the potential reduction of time and labor that one can expect by using exchangeable plates as opposed to direct feeding. Even though feed preparation using the conventional method takes approximately 50% less time than preparing the plates, the overall time needed to feed and clean could be reduced by nearly half, under the procedures used in this work.

A number of materials are employed as plates, and their practicality and durability make them economically efficient. Avicel (crystaline cellulose) has been successfully used in studies of gastropods (Sakata and Ina, 1992); corrugated plastic sheets have been also investigated (Shrestha and Knud-Hansen, 1994). In the present trial, acrylic proved effective, since food adhered firmly enough to last for 24 h and thus the feed lost to the bottom was negligible. However, as acrylic is expensive, it is important to experiment with cheaper, more porous materials at a larger scale.

In bullfrog tadpole culture, traditional management strategies used in order to overcome growth asynchrony include routine size-grading and separation (Lopes-Lima and Agostinho, 1992). This task demands a large number of holding tanks. Further research is needed to evaluate the effect of artificial grazing substrates in conditions of larger, commercial tanks, for if these plates improve the distribution of feed for tadpoles, they may help to reduce growth disparity, and consequently reduce the number of holding tanks needed.

Acknowledgements

We would like to express our gratitude to Drs Victor Vidal and Jack Frazier for their criticism and correction of the manuscript. We deeply appreciate the extremely enriching comments and assistance of one of the reviewers who anonymously helped improve the quality of this paper. The present study was supported by CONACYT-Mtxico, Project NO. 21 1085-5-1746A.

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