An evaluation of radiography in studies of gastric ... - Springer Link

Aquaculture International 6, 379–385 (1998)

An evaluation of radiography in studies of gastric evacuation in African catfish fingerlings Mostafa A.R. Hossain, Graham S. Haylor* and Malcolm C.M. Beveridge Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland

Groups of 3–4 g African catfish (Clarias gariepinus Burchell, 1822; Family: Clariidae) were offered feeds of two types in three combinations: feed with a radio-opaque marker (Ballotini, 0.16–0.25 mm), feed without marker and a 50:50 mixture of marked and unmarked feeds. Stomach contents were estimated by X-radiography and gastrectomy just after meal termination and at different time intervals (4, 8, 12, 16, 20, 24, 32, 40 and 48 h). There were no significant differences among treatment groups confirming that there was no effect of the marker on feed preference and evacuation rate. Estimations of gastric evacuation rate calculated from gastrectomy and by X-ray sampling were not significantly different. Therefore, gastric evacuation rate of African catfish can be accurately estimated by feeding marked feed and then using X-radiography to monitor feed passage through the gut. KEYWORDS: African catfish (Clarias gariepinus), Gastric evacuation, Radio-opaque marker

INTRODUCTION Feeding strategies in fish farming should be aimed at optimising growth and food conversion, and at minimising waste. Development of successful strategies may be aided by knowledge about food consumption patterns. Meal size and time between meals are important factors affecting daily food intake (Brett, 1979; Talbot and Higgins, 1983), so accurate measures of gastric evacuation may assist in estimating consumption, and have value for the development of feeding strategies. Food consumption by individual fish has been estimated in various ways, but Xradiography has several advantages. Fish can feed normally and the method is noninvasive. It is applicable to both small and large fish, food preparation is easy, and there is little associated risk to fish welfare (Talbot and Higgins, 1983). Although the method has been validated in feeding studies of Atlantic salmon, Salmo salar (Talbot and Higgins, 1983; Thorpe et al., 1990), Jørgensen and Jobling (1988) observed a difference in the evacuation of marker and nutritional content of feed in Arctic charr, Salvelinus alpinus. The method would thus appear to be species-specific to some extent and can only be used for evacuation studies when the marker moves through the gut at the same rate as the digesta. African catfishes (family Clariidae), especially Clarias gariepinus, are farmed in Africa and Asia (Kaiser et al., 1995), and with intensification of farming methods it is * Corresponding author. Tel: 0044–01786–465336; fax: 0044–01786–472133; email: [email protected] 0967–6120 © 1998 Chapman & Hall Ltd.

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important to be able to define feed requirements. The present study sets out to evaluate radio-opaque Ballotini as a marker to estimate gastric evacuation in African catfish. Specifically, two important questions are posed: (i) are there significant differences between estimates of feed ingested from X-radiographs of stomach contents of fish given feed with Ballotini and from gastrectomy and (ii) are there any effects of markers on feed preference and gastric evacuation? MATERIALS AND METHODS Fish C. gariepinus fingerlings of mean (SE) weight 0.95 (0.1) g, were obtained from broodstock maintained in the Institute of Aquaculture, University of Stirling. Larvae were fed with unhatched, de-cysted Artemia (Argent Chemical Laboratories, Redmond, USA) for a period of 4 days from 48 h after hatching and then weaned over a 4 day period by substituting Artemia with a commercial trout diet, ground and sieved to 250 m (Trouw Aquaculture UK Ltd). After weaning, larvae were fed on the ground trout diet for a further 16 days. Feed was dispensed continuously by belt feeder. Feed preparation A commercial pelleted trout diet (same diet used in larval rearing) was ground to a fine powder in a hammer mill and Ballotini glass beads (136–002, 0. 16–0.25 mm; Jencons Scientific) added at a concentration of 1% w/w. Some water was added to the mixture. After several hours mixing in a food mixer (Hobart A200) the feed was re-pelleted to a size of 2 mm (California Pellet Mill, Lab. Model CL2), the pellets freeze-dried, and then stored in sealed containers at 5 °C until use. A control diet without Ballotini was prepared in the same way. Samples of marked feed (n 5 28) of known weight (0.05–1.0 g) were X-rayed to establish the relationship between pellet weight and the number of Ballotini present: Y 5 0.00419 1 0.00209X; r2 5 0.99

(1)

Experimental procedure Seven hundred and fifty 26 day old C. gariepinus fingerlings were transferred to thirty 40 cm diameter rearing tanks within a recirculation system. Stocking density was 25 fish per tank. Tanks were covered by black polythene to reduce light levels. Water depth was maintained at 4 cm and shelter was provided. A 12 h light : 12 h dark regime (0830–2030 h, light period) was established and water temperature maintained at 30 (1) °C. Between day 26 and day 40, the fish were fed to apparent satiation, three times per day (0900, 1300, 1700 h). Each meal lasted approximately 10–12 min. At specific times during days 41–43, fish in 10 tanks were fed marked feed (Treatment A), 10 tanks were fed a 50:50 mixture of marked and unmarked feed (Treatment B) and fish in 10 tanks were fed unmarked feed (Treatment C). Fish were fed to apparent satiation and excess feed was removed after feeding ceased. Ten fish from each tank were then sampled at random at different time intervals following the termination of the meal (Table 1). All procedures were performed on anaesthetised fish. The first samples of fish were anaesthetised using 100 mg l–1 benzocaine solution, 5–10 min after they ceased

Gastric evacuation in African catfish

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TABLE 1. Feeding and sampling schedule on day 41–43 for African catfish fingerlings fed three different diets First Feeding Tank No. A1, A2, A3, A4, A5, A6, A7, A8, A9, A10,

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10

& & & & & & & & & &

Day 41 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10

Day 42

Day 43 09.00 09.00 09.00 09.00

17.00 17.00 17.00 09.00 17.00 13.00

Sampling time

Deprivation Period (h)

Day 43

0 4 8 12 16 20 24 32 40 48

09.00 13.00 17.00 21.00 09.00 13.00 17.00 17.00 09.00 13.00

feeding (handling or the application of anaesthetic 1–2 min after feeding was found to result in loss of ingested food). No losses of ingested feed were observed in any fish before or during X-raying. Fish were then weighed (Mettler PM6000 balance; precision 0.01g) and X-rayed. The stomach contents from each fish were then carefully removed and dried at 40 °C overnight. Stomach contents were then reweighed and calculated in terms of percent body weight. Further samples were taken at intervals (Table 1) and the changes in the amount of marked feed present in the stomach with increasing time was used to estimate gastric evacuation rate (GER). X-ray protocol Both the marked feed pellets and fish were X-rayed using a Machlett Aeromax 2 Xray apparatus (exposure time 2 s at 2 kV). Kodak Industrex film was used and the film developed using Kodak Industrex manual developer (4 min) and fixed by Kodak Industrex manual fixer (8 min) following washing (10 min) in cold, running tap water. The numbers of Ballotini were counted from X-ray plates observed under a binocular microscope (3 40 magnification) and the amount of feed was estimated from the calibration curve. Data analyses Stomach content was expressed in terms of per cent body weight: S5

Wf 3 100 W 2 Wf

(2)

where Wf 5 Weight of feed in stomach (g), W 5 Weight of fish (g). Stomach contents of fish fed the mixed diet were estimated by multiplying X-ray values by two. Regression analysis was done using the absolute value (g) of stomach content obtained from two methods (stomach contents from fish fed unmarked feed were not included in regression) and a significance test was performed on the regression coefficient. Finally the percent body weight data were arcsine transformed and then

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analysed by a series of one way ANOVAs on arcsine transformed data (Sokal and Rohlf, 1981). RESULTS AND DISCUSSION Highly significant correlations were found for the stomach content data obtained by both gastrectomy and the X-ray method (Fig. 1). In addition, a significance test on the regression coefficient (slope) indicated a highly significant (P , 0.05) positive relationship between the two sets of data, and the slope (0.928) did not differ significantly (P , 0.05) from 1. The stomach contents of fish immediately after feeding to satiation and after various deprivation periods are summarised in Table 2. The results show that average stomach contents at 0 h (feed consumption) ranged from 6.06 to 6.21% of body weight. The ANOVA performed on stomach content data obtained just after satiation prove that feed intake of fish fed three different diets are not significantly (P , 0.05) different, therefore, the marker, Ballotini has no effect on feed preference. The next seven ANOVAs performed on data obtained at different time intervals showed that the differences were not significant proving the marker had no effect on evacuation rate as well (Table 3).

FIG. 1. Relationships between stomach content (g) data obtained from gastrectomy and X-ray method. (Since the stomach contents of fish fed unmarked feed were obtained by gastrectomy only those data were not included in regression.)


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TABLE 2. Mean (95% confidence limit values) of stomach contents (% body weight) of fish in different treatments following different time intervals

Treatment

A Marked feed

Deprivation Period (h)

Weight from gastrectomy (g)

Estimated weight from Ballotini (g)


Estimated weight from Ballotini (g 3 2)


0 4 8 12 16 20 24 32

6.13 5.03 4.00 3.23 2.95 2.75 2.20 1.45

6.21 5.13 3.90 3.21 2.99 2.66 2.10 1.42

6.17 5.09 3.95 3.20 3.04 2.65 2.15 1.40

6.06 5.02 4.04 3.16 3.00 2.80 2.11 1.37

6.14 5.15 4.06 3.18 3.07 2.81 2.09 1.40

(0.16) (0.17) (0.27) (0.33) (0.41) (0.43) (0.33) (0.18)

B Mixed feed

(0.22) (0.38) (0.11) (0.52) (0.32) (0.28) (0.18) (0.14)

C Unmarked feed

(0.18) (0.33) (0.39) (0.41) (0.37) (0.47) (0.27) (0.12)

(0.38) (0.38) (0.42) (0.54) (0.47) (0.41) (0.22) (0.19)

(0.25) (0.20) (0.31) (0.39) (0.36) (0.24) (0.26) (0.17)

TABLE 3. One way ANOVA (Five groups with equal sample size) Ho : no difference in the quantity of marked feed (gastrectomy and X-ray method), unmarked feed (gastrectomy) or a 50:50 mixture (gastrectomy and X-ray method multiplied by 2) ingested after satiation (0 h) and different time intervals Deprivation

Sources Of Variation

SS

df

MS

F

Among Stomach Contents Within Treatments Total

0.12 7.45 7.57

4 45 49

0.03 0.17

0.18

0h


0.14 10.77 10.91

4 45 49

0.03 0.24

0.14

4h


0.17 11.92 12.08

4 45 49

0.04 0.26

0.16

8h


0.02 23.49 23.51

4 45 49

0.01 0.52

0.01

12 h


0.10 17.84 17.94

4 45 49

0.03 0.40

0.06

16 h


0.24 16.69 16.93

4 45 49

0.06 0.37

0.16

20 h


0.08 7.87 7.95

4 45 49

0.02 0.17

0.11

24 h


0.03 2.97 3.01

4 45 49

0.01 0.07

0.13

32 h

F(4,45) 0.05

2.58

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FIG. 2. Gastric evacuation of African catfish fingerlings. After 40 h there were no feeds in the stomachs. However, since it was not obvious at which exact point stomachs were completely evacuated, the last two points (40 and 48 h) were excluded from the evacuation rate calculation. Error bars are 95% CL. A, B and C are the treatment no. and represent marked, mixed and unmarked feed respectively and g and b express gastrectomy and Ballotini methods.

Fig. 2 shows the evacuation of pelleted feed from the stomach after various deprivation periods. The data can be described by the equation (Elliott and Persson, 1978): St 5 S0 e–Rt

(3)

where S0 5 stomach contents after first feeding to satiation, St 5 stomach contents after time t, R is gastric evacuation rate and t is time (h). It was not possible to determine the exact point at which all fish stomachs were


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fully emptied although this occurred after 32 h and before 40 h; hence, the last two points (40 and 48 h) were excluded from the regression. Gastric evacuation rates derived from five sets of data were not significantly different. CONCLUSIONS 1. The results indicated that the inclusion of Ballotini in diets fed to C. gariepinus has no effect on feed ingestion or gastric evacuation rate. 2. This is a useful and accurate method for estimating gastric evacuation and food intake in African catfish. ACKNOWLEDGEMENT The first author wishes to acknowledge the assistance of the British Council funding in carrying out this work. REFERENCES Brett, J.R. (1979) Environmental factors and growth. In: Fish Physiology (eds W.S. Hoar, D.J. Randall and J.R. Brett), Vol. VIII. Academic Press, New York, pp. 599–667. Elliot, J.M. and Persson, L. (1978) The estimation of daily rates of food consumption for fish. Journal of Animal Ecology 47, 977–991. Jørgensen, E. and Jobling, M. (1988) Use of radiography in feeding studies: a cautionary note. Journal of Fish Biology 32, 487–488. Kaiser, H., Weyl, O. and Hecht, T. (1995) The effect of stocking density on growth, survival and agonistic behaviour of African catfish. Aquaculture International 3, 217–225. Sokal, R.R. and Rohlf, F.J. (1981) Biometry, 2nd edn. Freeman and Company, New York. Talbot, C. and Higgins, P.J. (1983) A radiographic method for feeding studies using metallic iron powder as a marker. Journal of Fish Biology 23, 211–220. Thorpe, J.E., Talbot, C., Miles, M.S., Rawlings, C. and Keay, D.S. (1990) Food consumption in 24 hours by Atlantic salmon (Salmo salar L.) in a sea cage. Aquaculture 90, 41–47.