Camp. Biochem. Physiol. Vol. IOSA, No. 1, pp. 147-150, 1993 0300-9629193 $6.00 + 0.00 Printed in Great Britain Q 1593 Pergamon Press L&d

NO DIETARY VITAMIN B,, REQUIRED FOR JUVENILE TILAPIA URE~C~RU~~S MLOTKUS x 0. AUREUS

SHI-YEN SHIAU* and CHUN-QUI LUNG

Department of Marine Food Science, National Taiwan Ocean University, Keelung, Taiwan 202, R.O.C. (Tel. 02-462-2 192; Fax 02-462- I 684)

(Received 20 July 1992; accepted 28 August 1992)

Abstract-l. A I6-week experiment was conducted to evaluate the dietary vitamin B,, requirements for juvenile tilapia.

2. Six levels (0, 10, 20, 60, 100 and 2OO,ug/kg diet) of supplemental vitamin B,, were used. The folacin-free and folacin f vitamin B,, combined free diets were also included in the study for comparison.

3. Growth, hematological index and hepatosomatic index of tilapia were similar (P > 0.05) in all the dietary groups.

4. No pathological symptom was observed in fish fed either of the dietary groups throughout the experiment.

5. These data suggest that no supplemental vitamin B,, is required in diet for juvenile tilapia.

tNTR5DUffION

Vnamin Et,* is an essential growth factor in diets for most monogastric animals. Poor appetite, impaired hematopoiesis and poor growth are prominent signs of vitamin B,, deficiency in humans (Herbert, 1975), other warm-blooded animals (Oace and Chan, 1978) and Chinook salmon (Halver, 1957). Ruminant ani- mals do not require a dietary source of vitamin B,, because microorganisms in the digestive tract syn- thesize a sufficient quantity (Kon and Porter, 1954; Johnson et al., 1956). Hashimoto (1953) was unable to demonstrate dietary requirement for vitamin Bn for common carp, Cyprinus carpio, and later research demonstrate that bacteria from the gut of this species could synthesize vitamin Bn (Kashiwada and Teshima, 1966; Teshima and Kashiwada, 1967; Kashiwada et al., 1970).

Relatively little info~ation is known as to vitamin B,, requirements in tilapia. The dietary supplemen- tation level of vitamin B,, in the literature vary greatly. For example, 2Opg/kg (NRC, 1977) and 100 pg/kg diet of supplemental vitamin Bn (Jackson et al., 1982) are being used in studies. The purpose of the present investigation was to evaluate the dietary vitamin B,* requirements of tilapia, Oreochromis niioticus x 0. aurew.

MATERIALS AND METHODS

Diet preparation

Formulation of the experimental diets are given in Table 1. Vitamin free casein (38%, Sigma Chemical Co., St Louis, MO) and gelatin (2%, Sigma) were used as protein source. The vitamin mixture is similar -._ *To whom all correspondence should be addressed.

to that used by Shiau and Suen (1992) except that it did not contain vitamin B,,. Vitamin B,, (Sigma) was incorporated into the diet at 0, 10, 20, 60, 100, and 200pg/kg diet. Folacin free and (vitamin B,, f folacin)-free diets were also included as com- parison. The diets were prepared and stored as previously described (Shiau et al., 1987).

There was no observed difference in the palatability of diets: the time required for tilapia to complete a meal of any experimental diets was within 2min. There should have been no difference in leaching of water soluble nutrients between diets, because leaching from a similar diet was minimal after 2 min in water (Bordner et al., 1986).

Experimental procedure

Male tilapia, Oreochromis niloticus x 0. aureus, were supplied from the Far East Propagation Co. (Cha-Yi, Taiwan). After arrival, they were acclimated to laboratory conditions for 4 weeks in a plastic tank (74 w x 95 1 x 45 h cm) and fed a commercial diet (Lucky star, Hung Kuo Industrial Co. Taipei, Taiwan). At the beginning of the experiment 12 fish (mean weight: 1.03 rt 0.02 g) were stocked into each experimental aquarium (30.5 w x 61 .O 1 x 55.5 h cm). There were a total of eight treatments. Each exper- imental diet was fed to three aquaria of fish. Each aquarium was part of a close-recirculated system with a common water reservoir maintained at 26 &- 1°C. The water was circulated at 2 l/min through two separate biofilters to remove impurities and reduce ammonia concentrations. Dissolved oxygen con- centrations were at least 6ppm throughout the experiment.

The fish were fed 6% of their body weight per day in the first 10 weeks and 4% per day in the last 6 weeks. This amount was close for the maximum daily

147

148 SHI-YEN SHIAU and CHUN-QUI LUNG

Table I. Composition of experimental diets

Vitamin B,, content @g/kg diet)

Ingredient 0 IO 20 60 100 200

Vitamin-free casein 38 38 38 38 38 38 Gelatin 2 2 2 2 2 2 De&in 35 35 35 35 35 35 Corn oil 77 I I I I Cod liver oil 4 4 4 4 4 4 Vitamin mixture* (B,* free) I I I I 1 I Mineral mixture? 4 4 4 4 4 4 Alpha-cellulose I I I? I I CMC1 2 2 2 2 2 2 _ Analysed vitamin B,, (pg) 3.7 14.0 29.0 63.6 116.5 191.9

*Vitamin mixture: thiamin hydrochloride, 5 mg; riboflavin, 5 mg; calcium pantothenate, IO mg; nicotinic acid, 20 mg; biotin, 0.6 mg; pyridoxine hydrochloride, 4 mg; folic acid, I .5 mg; inos- ital. 200 mg; ascorbic acid, 100 mg; choline chloride, 400 mg; menadione, 4 mg; alpha-tocopherol acetate, 40 mg; para-amino benzoic acid, 5 mg; vitamin A acetate, 200 I.U. All ingredients were diluted with alpha-cellulose to I g.

tSalt No. 2+mineral premix No. 5 (NRC, 1973). Salt No. 2: calcium biphosphate, 13.58 g; calcium lactate, 32.7 g; ferric citrate, 2.97 g; magnesium phosphate (dibasic), 13.7 g; potassium phosphate, 23.98 g; sodium biphosphate, 8.72 g; sodium chlor- ide, 4.35g. Salt premix No. 5: salt mixture No. 2, IOOg; AICI, .6H,O, 0.015 g; KI, 0.015 g; C&I, 0.01 g; MnSO, H,O, 0.08 g; CoCl .6H,O, 0.1 g; ZnSO, .7H,O, 0.3 g.

fCMC: carboxymethylcellulose.

ration for tilapia according to the daily rations consumed by the tilapia with different size based on previous experience in our laboratory. The daily ration was subdivided into two equal feedings at 9 a.m. and 5 p.m. Fish were weighed once every 2 weeks and the daily ration adjusted accordingly. The light cycle was 11 hr (8 a.m.-7 p.m.) light/l3 hr (7 p.m.-8 a.m.) dark. The fish were fed the test diets for a 16 week period.

Growth as measured by the percentage body weight gain and feed conversion ratio (FCR) was calculated as described previously (Shiau et al., 1990).

The vitamin Bn was determined microbiologically, using the bacterium Lactobaciflus leichmannii (ATCC 7830) (AOAC, 1984). At the end of the feeding trial, fish were weighed, and six were selected randomly from each aquarium and blood, liver and feces were collected for vitamin B,, analysis. Blood samples were collected in heparinized capillary tubes from the caudal peduncle. The intestinal tract of the fish was removed and the fecal contents were stripped from the rectum portion. Plasma, separated from the blood by centrifugation, liver and feces were frozen and

stored at -20°C until analysed for vitamin B,,. Vitamin B,, content of the samples were determined microbiologically by the previously described pro- cedure for diet analysis.

The blood collected was also analysed for hemato- crit, hemoglobin, and erythrocyte count. Mean corpuscular volume (MCV) was calculated as (hem- atocrit/erythrocytes) x 100 and mean corpuscular hemoglobin concentration (MCHC) was calculated as (hemoglobin/hematocrit) x 100.

Statistical analysis

All data were analysed using one-way analysis of variance. Multiple comparisons among means were made with the Duncan’s New Multiple Range Test (Puri and Mullen, 1980). The aggregate type I error was set at 5% (P < 0.05) for each comparison.

RESULTS

The percentage weight gain, feed conversion ratio (FCR), and survival rate of tilapia fed diets with various levels of vitamin Bn are shown in Table 2. All these growth parameters were similar in fish irrespective of the diet consumed.

The hematological index and hepatosomatic index of tilapia fed diets containing various concentrations of vitamin B,, are shown in Table 3. Both hemato- logical index and hepatosomatic index in tilapia were not significantly different among all the dietary groups.

The vitamin B,, concentration in liver, plasma, and feces of tilapia fed different diets are presented in Table 4. The liver vitamin B,, concentrations in tilapia fed different diets can be divided into three overlapping clusters. Low in fish fed 0 and 10 ,ug/kg diet, intermediate in fish fed 20, 60, and lOOpg/kg diet and high in fish fed 200 pg/kg diet. The difference between the low and the high groups were significant (P < 0.05). The plasma B,, concentration in fish fed the 100 and 200pg/kg diets were significantly (P < 0.05) higher than fish fed 0, 10, and 20 pg/kg diets. The fecal vitamin B,, concentration in fish fed 60 and 200 pggikg diets were significantly (P < 0.05) higher than fish fed 0, 10 and 20 pg/kg diets.

Table 2. Weight gain, feed conversion ratio (FCR) and survival rate of tilapia fed diets containing various levels of vitamin B,, for I6 weeks’

Vitamin B,, level. uR/kR

Initial body wt (a)

Final body wt (R)

Weight gain (%) FCR

Survival rate (%)

0 IO 20 60

100 200 Folacin-freet (B,2 + Folacin)-free Significance

I .02 + 0.02 1.04 f 0.01 1.04 + 0.01 1.03 ?r 0.02 1.03 fO.01 I .03 f 0.02 I .03 f 0.01 I .06 ? 0.02

NS

42.71 + 4.25 46.50 + 5.79 47.72 + 6.49 44.85 + 5.59 43.42 f 3.89 42.91 f 5.81 41.27 + 4.31 42.03 k 5. I I

NS

4050.00 * 470.99 4343.64 & 575.23 4496.52 + 654. IO 4081.49 f 763. I2 4120.00 + 418.29 4040.00 ? 589.54 3898.39 f 423.84 3942.47 f 503.04

NS

0.96 f 0.12 I .02 * 0. I2 I .04 rt 0.07 0.87 f 0.04 0.91 * 0.13 1.05 i 0.15 0.98 +0.16 1.07 * 0.01

NS

83.33 + 16.67 83.33 f 14.43 91.67 f 14.43 12.22 * 17.34 75.00 * 14.43 86.1 I + 4.82 88.89 f 12.73 72.22 + 9.62

NS

‘Values are mean f SD; N = 12. tFolacin-free: contains lOOrg/kg of vitamin B,, NS: insignificantly different at P > 0.05.

Juvenile tilapia do not require supplemental B,, 149

Table 3. Hematological index and hepatosomatic index of tilapia fed diets containing various levels of vitamin B,, for 16 weeks’

Vitamin B,, level, pgikg

Hematocrit (%)

Hemoglobin Erythrocytes (g/dt) (106/mm’)

MCVt (mm’)

MCHC: Hepatosomatic (%) index f%)

0 10 20 60

100 200 Folacin-free$ (B,2 + Folacin)-free

29.07 + I .25 6.84 f 0.63 I .76 & 0.32 29.07 f 0.87 6.70 + 0.50 1.64 i: 0.26 28.40 it 0.73 6.65 k 0.27 1.77 * 0.41 29.iO F 1.43 6.57 f 0.35 1.59 + 0.06 27.17 F 0.48 6.51 ? 0.51 I.71 + 0.21 30.00 * I .28 6.86*0.14 1.62 & 0.10 28.16 k 1.00 6.37 ? 0.40 I.71 rt 0.20 26.83 i I. I3 6.98 + 0.23 1.36 k 0.11

NS NS NS

171.29 + 37.45 40.26 t 8.50 1.97&0.10 181.66 + 29.67 4 1.45 f 4.29 1.90+0.1i 170.69 + 42.72 39.84 f 9.51 1.76+0.13 182.86 + 15.41 41.24 f 3.09 1.84 i 1.10 161.12 + 20.77 38.76 f 6.69 I .73 + 0.26 186.27 + 18.46 42.52 k 2.80 1.85+0.19 167.82 + 26.51 37.49 i 2.27 1.77 + 0.22 197.87 + 20.28 51.49 rt 5.33 I .77 + 0.32

NS NS NS

“Values are mean 2 SD; N = 12. tMCV: mean corpuscular volume. fMCHC: mean corpuscular hemoglobin concentration. QFolacin-free: contains 100 pg/kg of vitamin B,,. NS: insignificantly different at P > 0.05 in each column.

DISCUSSION

‘st has been reported that channel catfish showed vit.lmin B,, deficiency signs fed diets deficient in vitrmin B,, when their weight gain had increased by as much as eight-fold (Dupree, 1966). In the present study, after 16 weeks, approximately a 40-fold in- crease in weight gain was observed in fish fed all the experimental diets and none of the fish was showing deficiency symptoms. Such a growth should have depleted the fish of body-stored vitamin B,, if they were not absorbing an adequate amount from the digestive tract. A comparable liver vitamin B,, con- certration in fish fed diet without vitamin B,, sup- plementation to those fed diets with the vitamin B,, supplementation up to 100 pig/kg (Table 4) suggest that fish in none of the treatments were deficient in vimmin B,,.

It generally has been believed that intestinal micro- organisms contribute vitamin B,, to the host. Love11 and Limsuwan (1982) have demonstrated that tilapia, 0. nifoticus synthesized at least 11.2 ng of vitamin B,:‘g body wt/day. It remains unclear, however, as to whether the body synthesized vitamin B,, by tilapia car; meet its requirement because in their study only onr: concentration of 20pg/kg of vitamin B,, was incorporated into the basal diet and compared to the control diet (without the vitamin B,, supplemen- tation). In the present study, with various vitamin B,, supplementation levels in the diet, our results demon- strated that dietary supplementation of vitamin B,, is not necessary for normal growth of juvenile tilapia, Owochromis niloticus x 0. aweus.

Table 4. Vitamin B,, concentration in liver, plasma and feces of tilapia fed various diets*? -

Vitamm B,, Liver Plasma Feces Ieve!, pg/kg (pgig) (ngiml) (cl gig)

0 289.56 + 56.56* I .50 f 0.79’ 715.97 f 154.73’ IO 280.43 I87.51” I .70 f 0.90” 736.54 k 80.52” 20 293.19 + 25.49’b 2.46 + 1 .04ab 796.37 k 52.2@ 60 303.84 f 55.8vb 3.43 z 0.61& 998.64 F 180.3@

100 375.24 + 80.1 3Yb 4.43 + 0.57c 872.95 + 140.Wb 200 464.34 rt I 13.47b 3.93 k 0.4ge 1051.76; 152.4?

*Values are mean + SD; N = 12. tFigures in each column having the same letter were not significantly

different (P > 0.05).

There are still many unanswered questions regard- ing the availability of synthesized vitamin B,, to the host. These include questions of the localization of the bacteria which synthesize the vitamin and the ability of absorption of the synthesized vitamin in fish. Colon microfloria is one of the most complex and concentrated populations of bacteria found in nature. Callender and Spray (1962) delineated that human colon bacteria make large amount of vitamin Bn. However, the synthesized vitamin B,, is not absorbed through the colon wall, thus is not available to the non-coprophagous animal. In the present study, although the actual absorption rate of vitamin B,, was not measured, a rather high plasma vitamin B,, concentration was observed. Love11 and Limsuwan (1982) have reported that the plasma .

vitamm B,, concentration of channel catfish fed basal diet contained 20pg/kg of vitamin B,, was 0.91 ng/ml. In our study, I .5 ng/ml of plasma vitamin B,, concentration was obtained in tilapia fed diet without the vitamin B,z supplementation and the plasma vitamin concentration generally increased as the dietary vitamin incorporation level increased (Table 4). The high plasma vitamin B,, concentration in tilapia may suggest that the absorption of the vitamin from the intestine of this species. The liver vitamin B,: concentration in tilapia fed different experimental diets generally followed the plasma concentration pattern. These may provide an expla- nation of the similar growth performance of tilapia fed all the dietary groups.

The other possible route of absorption of vitamin Bn by the fish, discounting coprophagy, is gill ab- sorption from the aquarium water. Limsuwan and Love11 (1981) speculated that less than O.O2pg/l of vitamin B,, leached out from channel catfish feces into the aquarium water and suggested that gill absorption of this vitamin at this low concentration seems doubtful.

Very little is known on the combined effect of vitamin B,, and folacin deficiencies in fish. It has been reported that the combined deficiency of vitamin B,, and folacin resulted in a more pronounced anemia in l;abeo rohita Ham suggesting that folacin and

1.50 SHI-YEN SHIAU and CHUN-QUI LUNG

vitamin B,, have a supplementary and complemen- tary role in fish metabolism (John and Mahajan, 1979). In the present study, fish given either vitamin B,, free, folacin free, or both vitamin B,, and folacin free diet, the growth performance, hematological index and hepatosomatic index were all similar to those fed diets supplemented with various levels of vitamin B,,. Further study with gradient folacin levels in the diet may be needed.

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