Partha Bandyopadhyay Æ Pradeep K. Das Mohapatra
Materials and methods
estimated by micro-Kjeldahl method. Crude lipid contents were determined by the soxhlet extraction
method using petroleum ether (boiling point: 40–60 C) in the electro-thermal Soxhlet apparatus. After extraction of lipid, the defattend samples were used for the estimation of crude fiber following Patra (2002). Ash content was estimated by incinerating samples in a muffle furnace at 500 ± 50 C for 10 h. One hour after feeding, the leftover feed was initially siphoned out and an equal amount of water replenished. For fecal matter analyses, pooled fecal matter was collected into Petri dishes from the bottom of the aquarium every 2 h by the help of a pipette (Singh 1989). The collected material was stored at -20 C (Sundaryono et al. 1996). The collected material was dried in an oven (M/s Modern Industrial) at 55 C, ground and preserved in airtight containers.
Biochemical analysesDNA (deoxy-ribo nucleic acid) and RNA (ribonucleic acid) contents in 200 mg of liver (hepatopancreas) tissues were estimated as per the scheme given by Munro and Fleck (1969). The activity of the two digestive enzymes, protease and a-amylase, in the intestine of the fish were determined according to the method of Bernfeld (1955) as modified by Snell and Snell (1971). The GOT and GPT activity in the liver were determined following the method of Bernfeld (1955), while ACP activity was determined the method of Bramley (1974) and ALP activity by Rosauki (1993).
Determination of immunity levelsOn day 60, blood was collected from fish of each group. Part of the blood was heparinized and the rest was allowed to clot for serum samples, which were preserved at -20 C for further analysis. Immediately after collection, the heparinized blood samples of each group were pooled to three aliquots. Part of the blood was analyzed for leucocrit value in duplicate per sample (Blaxhall and Daisley 1973). The rest of the heparinized blood was immediately used for the phagocytic assay (Siwicki et al. 1994; Park and Jeong 1996).
Challenge trialAfter feeding for 60 days, the fish in each treatment were challenged with Aeromonas hydrophila AH1, which had been cultured and maintained in the Aeromonas selective medium (M884, Hi-Media). Fish in all replicates were immersed in a suspension of Aeromonas hydrophila AH1, *105 c.f.u. ml-1 according to Austin et al. (1995). This was followed by a second immersion *107 c.f.u. ml-1 after 7 days (Austin et al. 1995).
Statistical analysisAs all the above analyses were carried out on pooled samples of a given lot, standard errors or standard deviation of means were calculated. However, for evaluating the dietary performances, nutritional indices, enzymatic activities, and RNA:DNA ratio, immunological parameters, and challenge trials, significant differences between the means of the treatments were tested by Duncan Multiple Range Test (Duncan 1955) through SAS (1991).
ResultsAll the four experimental feeds (CC, C1, C2, and C3 in Table 1) were almost isocaloric and isonitrogenous. The average crude protein percentage on dry matter basis was around 36.92 and the gross energy was around 17.08 kJ g-1 (Table 2). The growth of Catla catla in relation to various
feeds was presented in Fig. 1. Significantly (P B 0.05) highest growth (20.15 ± 0.075 g) was obtained from fish fed feed C2, whereas lowest (16.95 ± 0.120 g) occurred in fish fed feed C3, which was even lower than the control (feed CC). Fish fed feed C2 showed significantly (P B 0.05) highest live weight gain (14.00 ± 0.086 g) followed by C1, CC, and C3 (Table 3). Significantly (P B 0.05) lowest FCR (2.26 ± 0.021) was obtained
from fish fed feed C2 while the highest (3.30 ± 0.042) was recorded in case of fish fed feed C3 (Table 3). It was also observed that significantly (P B 0.05) highest SGR of 1.98 ± 0.011 was obtained in the fish fed feed C2 showing better utilization of nutrients and the lowest (1.55 ± 0.014) in fish fed feed C3 showing lowest utilization of nutrients. Similarly, the highest (P B 0.05) PER of 1.22 ± 0.010 was obtained in fish fed feed C2, which indicated better utilization of protein for growth and metabolism (Table 3). Initial and final carcass composition of Catla catla in relation to various feeds was presented in Table 4. The carcass composition of the fish revealed an apparent increase in the final carcass protein and lipid (P B 0.05) over the initial carcass protein and lipid. Significantly (P B 0.05) highest carcass protein (65.48 ± 0.002%) and lipid (22.80 ± 0.004%) was recorded in fish fed feed C2, while the lowest was in the controls (CC). These results indicated that enhancement of carcass quality by probiotic-supplemented (Bacillus circulans PB 7) feeds may be due to enzymatic activity in the gut and thereby better nutrient utilization. Proximate composition of fecal matter of Catla catla during the 60-day feeding trial is presented in Table 5. Fecal matter proximate analysis revealed significantly (P B 0.05) greatest nitrogen excretion (14.86 ± 0.006%) in fish fed feed CC and least (11.52 ± 0.007%) in fish fed feed C2 (Table 5). The crude lipid remained between 02.19 ± 0.001% (feed C1) and 3.16 ± 0.002% (feed CC). The relationships of different water quality parameters did not follow any specific trend of controlled conditions and isocaloric feeds. The water quality during the study, period remained in the following
ranges: pH, 7.47 ± 0.182–7.52 ± 0.164; total alkalinity 130.80 ± 4.980–140.25 ± 9.680; DO, 4.40 ±
0.595–5.63 ± 0.420; total ammonia, 0.270 ± 0.031– 0.390 ± 0.028, and average temperature was
30.00 ± 1.155. Significantly (P B 0.05) greater RNA:DNA ratio (1.95 ± 0.011) was registered in fish fed feed C2 and least (1.66 ± 0.010) in fish fed feed CC (Fig. 2). It was also observed that the RNA:DNA ratio of fish increased in all the treatments over the initial RNA:DNA ratio. Significantly (P B 0.05) highest protease was recorded in fish fed feed C2 (47.9 ± 0.016) and lowest in fish fed feed C3 (32.10 ± 0.009) (Table 6), whereas there were no significant differences (P B 0.05) in a amylase activity (Table 7). The ACP activity in the liver was greatest (P B 0.05) in case of fish fed feed C2 (3.78 ± 0.027) and least (2.06 ± 0.032) in case of fish fed feed CC (Table 8). Similarly, ALP activity in the liver was shown a similar trend (Table 8).
Significantly (P B 0.05) highest GOT (0.052 ± 0.000) and GPT (0.083 ± 0.002) values were registered in fish fed feed C2, whereas lowest (0.045 ± 0.000) GOT value was recorded in fish fed feed CC and GPT value (0.044 ± 0.005) in fish fed feed C3 (Table 8).
The effect of different feed treatments on the albumin:globulin ratio is presented in Fig. 3. Highest
albumin:globulin ratio was recorded in fish fed feed CC and lowest in fish fed feed C2. The albumin:globulin ratio showed a similar trend after challenge trial, but the values were slightly lower than the feeding trial, perhaps because of decrease in TSP values. The effect of probiotics (Bacillus circulans PB 7) on non-specific immunity was observed. Significantly (P B 0.05) highest phagocytic ratio (63.00 ± 1.22), phagocytic index (2.34 ± 0.09), and leucocrit value (62.20 ± 1.80) were recorded in fish fed feed C2, whereas the lowest of all the values (8.00 ± .086, 1.49 ± 0.20, and 34.10 ± 3.86, respectively, were recorded in fish fed feed C3 (Figs. 4, 5, and 6, respectively). After a 10-day challenge trial, significantly (P B 0.05) highest survivability (96.66%) was
observed in fish fed feed C2, followed by fish fed C1 (53.33%), C3 (40%), and CC (6.66%) (Fig. 7).
Regression results of different parameters with respect to different probiotic supplements in Catla
catla are presented in Table 9.
Aerobic gram-positive endospore-forming bacteria, i.e., Bacillus sp., have been evaluated as probiotics, with uses including the improvement of water quality
by influencing the composition of water-born microbial populations and by reducing the number of
pathogens in the vicinity of the farmed species (Wang et al. 1999). Thus, the bacilli are thought to
antagonize potential pathogens in the aquatic environments. This is curious because it is generally
accepted that laboratory cultures do not survive well when re-introduced into the natural environment, the cells being often outcompeted/antagonized by the natural microflora (Austin et al. 1995). Nevertheless, direct benefits from the use of the bacilli were a reduction in the use of chemicals in the aquatic environment and enhanced growth of farmed species (Wang et al. 1999).
The use of probiotics has been accompanied by a concomitant reduction in the levels of antimicrobial
compounds (particularly antibiotics) used in aquaculture, and improved appetite and/or growth
performance of the farmed species. The former is obvious insofar as if the animals are otherwise
healthy then there will no need to use antimicrobial compounds. However, the inference about improved appetite and growth is more difficult to reconcile. In particular, it is important to determine whether or not probiotic actually tastes good or does it modify the feed thereby improving digestibility (and taste). Apart from laboratory preparations of bacteria, some workers have used commercially available products. For example, Queiroz and Boyd (1998)
and Moriarty (1998) used commercial preparations containing Bacillus sp. in catfish and shrimp ponds, respectively. Hirata et al. (1998) used mixed cultures consisting mainly of Bacillus sp. to improve the performance of the rotifer, Branchionus plicatilis, in water. Furthermore, Kennedy et al. (1998) used Bacillus 48 to enhance the quality and viability of common snook, Centropomus undecimalis (Bloch). These workers found that Bacillus improved the survival of larvae, increased food absorption by enhancing protease levels, and gave better growth. Also, the probiotic decreased the number of suspected pathogenic bacteria in the gut. It is noteworthy that Chang and Liu (2002) used Bacillus toyoi and Enterococcus faecium SF 68 from commercial products to reduce Edwardsiellosis in the European eel, Anguilla anguilla (L.). An extracellular proteaseproducing bacteria, Bacillus circulans (Lr 1.1), was isolated by Ghosh et al. (2003) from the gut of Labeo rohita fingerlings and used as supplement in the diets
and the effect of supplement on growth performance and utilization efficiency of L. rohita was measured. The diet containing 1.5 9 105 Bacillus circulans cells per 100 g showed significantly better growth, lower feed conversion ratio, higher protein efficiency ratio, and highest protease activity. Similar observations were also recorded in the present study, where 2 9 105 Bacillus circulans PB 7 cells per 100 g showed significantly better growth, lower feed conversion ratio, higher protein efficiency ratio, and highest protease activity. In this study, although all the feeds were isonitrogenous, the concentration of probiotics in the C2 feed might be helpful for proper nutrient utilization. The whole body carcass composition and lower nitrogen egestion which were observed in fish fed feed C2 is attributable to proper probiotic concentration, whereas reduced carcass composition and greater nitrogen egestion were observed in fish fed feed C3, which could have also been due to the overall low feed utilization level. RNA:DNA is known to provide a dependable indication of growth trend (Buckley 1980; Khan and Jafri 1991; Bandyopadhyay et al. 2005). The ratio was the greatest in the fish fed C2 feed with higher dietary utilization and best growth. Bazaz and Keshavanath (1993) found a higher RNA:DNA ratio in better growing fish fed with oil-supplemented diets using equal level of crude protein. The present study also reports such a finding where all the feeds were isocaloric and isonitrogenous, but 2 9 105 Bacillus circulans PB 7 cells per 100 g incorporated feed (feed C2) exhibited better growth as well as better RNA:DNA ratio. The highest levels of GOT and GPT were found with the feed C2 and the lowest in the control (feed CC),
which may be because of the better dietary protein utilization. Most of the amino acids normally found in protein undergo transamination reactions, and transaminases are localized in both cytosol and mitochondria (Wada and Marino 1964), which is induced by high protein diet (Swick et al. 1965). Thus, a positive correlation between the probiotic concentration and the GOT and GPT levels in the liver could be observed. The ALP and ACP activities of the liver also showed an excellent trend and were dependent upon the probiotic concentration in the feed. It was observed that the probiotic-containing feed C2 showed higher ALP and ACP activity as well as higher dietary utilization and growth, which was similar to the observation of Bandyopadhyay et al.
(2005). In this present study, a superior growth performance in terms of live weight gain and specific
growth rate was noted in C. catla fed feed C2 when compared to other treatments. This observation draws attention to an essential inference that the probiotic concentration, which was used in this feed, might be helpful for optimum dietary utilization. It was also observed that P/E ratio was highest in fish fed feed C2 although with the highest dietary utilization. Mohanty et al. (1996) reported that the higher the dietary utilization the higher the P/E ratio, which was contradicted in fish fed feed C2 in this study. This may be because of the supply of higher energy from the higher carcass lipid content in fish fed feed C2. The results obtained in this study not only support the use of probiotic (Bacillus circulans PB 7) for better growth and proper nutrient utilization but also confirm it to be an important immunostimulant in Catla catla. There was a statistically significant (P B 0.05) increase in all the parameters of non-specific immunity examined, as well as a higher survival against the pathogenic Aeromonas hydrophila infection, thus indicating it as a potent immunostimulant in Catla catla. Highest PR, PI, and leucocrit value (P B 0.05) and lowest albumin: globulin ratio (P B 0.05) were observed in fish fed feed C2 might be due to increased lymphocyte proliferation and subsequent immunoglobulin production. A similar observation was made by Sahoo and Mukherjee (2001) by using b-1,3, glucan as an immunostimulant. The findings of this study suggest that the concentration of probiotic Bacillus circulans PB 7 applied in feed C2 was able to increase the overall physiological performances and enhance the defense mechanism in the fingerlings of Catla catla. Further investigation should be applied by preparing the feeds with different concentrations of this probiotic bacterium between the two best feeds achieved in this study. The findings should also be confirmed in outdoor, earthen pond, trials before commercialization.