Preliminary Evidence of the Contribution of the Intestinal Microflora to Biotin Supply in Zebrafish Danio rerio (Hamilton-Buchanan)

Abstract

A study was conducted to preliminarily assess the contribution of the intestinal microflora to biotin supply in zebrafish. Biotin and avidin were added to three isonitrogenous and isocaloric purified diets to provide molar avidin: biotin ratios of 0:0 (basal diet), 0:1 (biotin-supplemented diet), and 120:0. Another diet was made by supplementing the antibiotic succinylsulfathiazole (1%, wt/wt) to the basal diet. A fifth diet was the Zeigler commercial diet for zebrafish. Each diet was fed to a triplicate group of fish (mean initial mass 0.266 g) for 8 weeks. The condition factor, feed conversion ratio (FCR), percentage weight gain, and survival were similar in fish groups fed the commercial and the biotin-supplemented diets, but energy conversion efficiency and whole-body biotin content were highest in the fish fed the commercial diet (p<0.05). Reduced growth and survival, and increased FCR were noted in fish fed basal diet compared with those fed biotin-supplemented diet. The supplementation of avidin in diet led to lower survival and condition factor, and higher FCR than that observed with basal diet. Intestinal microbial synthesis is assumed to be a significant source of biotin to the zebrafish, as fish fed the antibiotic-supplemented diet showed the lowest growth, health condition, and feed utilization.

Introduction

The zebrafish, Danio rerio (Hamilton-Buchanan), is a small cyprinid widely used as a model organism in studies of developmental biology, physiology, molecular genetics, toxicology,^1–4 and reproductive biology.⁵ The zebrafish has also attracted increasing interest recently as an animal model for human disease research and screening of human therapeutics.^6,7 A number of favorable attributes, including its small size, rapid development and generation time, prolific spawning rates, the ease of handling follicular cells under laboratory conditions, optical transparency during early development, tractability in genetic screens, and genetic similarity to humans, have fueled its rise in popularity as a living model for biological studies and will likely continue to spur scientific interest.^3–5 Despite such popularity, only a few nutritional studies without live food have been conducted on this fish species. Outcomes of these studies demonstrate that zebrafish larvae can be reared without live food with substantial growth rates and high survival rates, if an appropriate artificial diet is continuously provided.² Feeding adult zebrafish flake diets to satiation thrice daily leads to maximum production of viable offspring, and the success achieved with laboratory-prepared diets can establish the foundation for an open-formulation nutritional standard that does not generate research results confounded by nutritional vagaries.⁴ However, the nutrient requirements of zebrafish (proteins/amino acids, lipids, carbohydrates, minerals, and some vitamins), at any life stage, are yet to be completely quantified.^3,4,7,8 This information should be known to be able to design properly adequate diets and feeding protocols for zebrafish.³ Of recent interest is biotin, a water-soluble B-complex vitamin, which is an essential vitamin for fish in general,^9,10 and zebrafish in particular.¹¹ Biotin serves as a covalently bound coenzyme for five carboxylases: acetyl-CoA carboxylase (isoforms alpha and beta; E.C. 6.4.1.2), pyruvate carboxylase (E.C.6.4.1.1), propionyl-CoA carboxylase (E.C. 6.4.1.3), and 3-methylcrotonyl-CoA carboxylase (E.C. 6.4.1.4).^9,12–15 An increasing body of evidence indicates that biotin also functions as a regulator of gene expression, likely via covalent modification of specific lysine residues in histones.^15–18 Biotin was discovered in the search for a nutritional factor that prevents egg white injury in experimental animals exposed to the biotin-binding protein avidin.¹⁹ Avidin is a water-soluble, basic glycoprotein found in raw egg white.^20,21 Avidin is a homologous tetramer, each 128-amino acid subunit of which binds a molecule of biotin apparently by linking to four tryptophan residues and an adjacent lysine in subunit binding site. The binding of biotin to avidin is the strongest noncovalent bond in nature, with a dissociation constant of ∼10⁻¹⁵ M.^20,21 The supplementation of raw egg white or avidin to diets induced biotin-deficiency signs in some mammalian species such as cats, hamsters, pigs, and rats as well as in several fish species.^13,22–24 Signs of biotin deficiency in fish include reduced growth, increased mortality, abnormalities in skin, intestine, and gill tissue (including blue slime patch disease), lesions of the colon, reduced food consumption, and convulsions.^{9,11,13,22–31} Reduced activity of biotin-dependent enzymes and an accumulation of hepatic glycogen are also well documented in biotin deficiency in fish in general.^9,11,13,32 The dietary biotin requirement for zebrafish growth was estimated at 0.51 mg kg⁻¹ diet.¹¹ On the other hand, feeding zebrafish a diet with 60 to 120 times more avidin than the dietary biotin requirement level (on molar basis) will cause biotin deficiency signs.²⁴ Despite the severe biotin deficiency signs observed in these two last studies,^11,24 the mortality of zebrafish remained more than 70% during a 12-week feeding period. A reason evocated was additional biotin supply by the intestinal microflora of zebrafish, as it is generally known that microorganisms indigenous to the intestinal tract of many freshwater fish species could synthesize and supply in good quantity biotin to the host.³³ On the other hand, it was assumed that the purified diets used in these studies,^2,11,24 contained a high amount of dextrin (23%), which is known to strongly favor the intestinal production of biotin in mature animals, as was the case in fowls,³⁴ although it is not yet demonstrated in fish. Nevertheless, it is well established that the addition of the antibiotic succinylsulfathiazole (1%, wt/wt) in fish diet suppresses intestinal synthesis of vitamins by the microflora.^35–39 The hypothesis of the current study is that the supplementation of antibiotic in the diet, to suppress the intestinal microflora, leads to lower growth, condition and whole-body biotin content, and higher mortality of zebrafish. This study was, therefore, designed to present preliminary evidence of the contribution of the intestinal microflora to biotin supply in the model organism, zebrafish.

Materials and Methods

The experimental protocol was conducted in accordance with the guidelines of the Canadian Council on Animal Care, and with the authorization of the Animal Protection Committee of Université Laval.

Experimental diets

The basal diet used in this study was the purified diet proposed by Carvalho et al.² This diet was formulated by using vitamin-free casein as protein source. This purified diet has been shown to be adequate for zebrafish,² and was similar to that used by Yossa et al.^11,24 When biotin was added to a diet, the quantity was equivalent to the biotin requirements of zebrafish, which was estimated at 0.51 mg biotin kg⁻¹.¹¹ Three diets were formulated by adding biotin and avidin to the basal diet, at the molar avidin:biotin ratios of 0:0 (A0B0, basal diet), 0:1 (A0B1, biotin-sufficient diet), and 120:0 (A120B0, biotin-deficient diet), and the amount of cellulose in the vitamin mix was reduced to compensate (Table 1). A fourth diet was made by supplementing the antibiotic succinylsulfathiazole (1%, wt/wt) to the basal diet (A0B0S), and was referred to as the negative control (Table 1). Addition of this antibiotic was designed to suppress intestinal synthesis of vitamins.^35–39 The composition of these diets is presented in Table 1. A fifth diet in this experiment (ZC diet) was the Zeigler commercial diet for zebrafish (Adult Zebrafish Diet by Zeigler^®, Aquatic Habitats™), and was referred to as the positive control.

Table 1.

Composition of the Seven Experimental Diets Fed to Zebrafish Danio rerio During 8 Weeks

	Experimental diets ^b
Ingredients (for 1000 g diet) ^a	ZC diet	A0B0	A0B1	A120B0	A0B0S
Casein		450	450	450	450
Sodium casein		113	113	113	113
Casein hydolysate		37	37	37	37
Peanut oil		30	30	30	30
Cod liver oil		10	10	10	10
Phospahtidylcholine		20	20	20	20
Choline chloride (50%)		10	10	10	10
Dextrin		230	230	230	230
Mineral mix^c		50	50	50	50
Vitamin mix (biotin-free)^d		50.000	49.999	33.466	40.000
Biotin		0	0.00051	0	0
Avidin		0	0	16.533	0
Succinylsufathiazole		0	0	0	10
TOTAL (g)		1000	1000	1000	1000
Proximate composition
DM (%)	91.06	88.58	88.62	88.33	88.55
Ash (% DM)	3.13	2.15	2.16	2.15	2.07
Crude protein (% DM)	64.69	58.81	58.01	58.89	59.60
Crude lipid (% DM)	7.10	6.00	6.05	6.09	6.15
Energy (kcal g⁻¹ DM)	6.47	5.47	5.48	5.49	5.86
Biotin concentration upon analysis (mg kg⁻¹ diet)	4.9	0.062	0.486	0.073	0.094

Casein (vit–free), sodium casein, choline chloride and biotin were from MP biomedicals, LLC; Casein hydrolysate (C0626) Succinylsufathiazole (S8752) and Dextrin (D2256) were from Sigma; Peanut oil was from Selection Merit, Metro; Cod liver oil (19567) was from Swiss Herbal Remedies Ltd; Phospahtidylcholine (61755) was from Fluka; Avidin was from Neova Tecnologies Inc.

A0B0, A0B1, and A120B0 refer to the basal purified diets where the molar avidin:biotin ratio's added are 0:0, 0:1, and 120:0 respectively, while A0B0S refers to the basal diet containing the antibiotic succinylsulfathiazole (1%, wt/wt) with neither biotin nor avidin supplementation; ZC diet refers to Zeigler commercial diet for zebrafish (Adult Zebrafish Diet by Zeigler^®, Aquatic Habitats™).

Per kilogram of diet: cobalt sulphate.7H₂O, 0.002 g; copper sulphate.7H₂O, 0.025 g; iron carbonate, 0.2 g; potassium, iodide, 0.003 g; magnesium oxide, 2.5 g; manganese oxide, 0.1 g; sodium selenite, 0.0015 g; and the filler (alpha-cellulose).

Per kilogram of diet: retinol, 90,000 IU; calciferol, 10,000 IU; α tocopherol, 0.175 g; ascorbic acid, 0.25 g; thiamin-HCl, 0.075 g; ribofavin, 0.125 g; Ca pantothenate, 0.25 g; nicotinic acid, 1 g; pyridoxine-HCl, 0.025 g; folic acid, 0.05g; cyanocobalamin, 0.1 g; menadion sodium bis., 0.05 g; inositol, 2 g; and the filler (alpha-cellulose); biotin and avidin were added to the vitamin mix at the expense of alpha-cellulose.

DM, dry matter.

The diets were prepared by thoroughly mixing the ingredients (except biotin and avidin) with oil. Solutions of dissolved biotin and avidin in cold water (45% vol:mass of the total dry weight of the other ingredients) were then added until they resulted in a stiff dough. This was then passed through an extruder with dye, and the resulting spaghettis were cut in small pieces (about 2 mm) and dried in a forced air oven at 28°C for 12 h. After drying, the diets were broken further, sieved into convenient pellet size (0.5–0.8 mm), and stored at −20°C. All the diets were analyzed for proximate composition,⁴⁰ and biotin content was analyzed by competitive enzyme-linked immunosorbent assay (ELISA)^41–44 (Table 1). To determine the biotin content of the diets, 1 g of each diet was extracted into 9 g of assay buffer (0.1 M HEPES, 1 M NaCl, 0.05% Tween 20, and pH 7.0) to produce a 1:10, g g⁻¹ dilution. Diet was mixed for 1 h, and then centrifuged to pellet non-soluble material. An aliquot was sampled from between the floating lipid layer and the pellet. This light yellow aqueous extract layer was diluted by 50-fold, and biotin was measured by competitive ELISA. The dry matter (DM), ash, crude protein, crude lipid, gross energy, and analyzed biotin content of the experimental diets are presented in Table 1.

Fish, facilities, and feeding

Healthy zebrafish juveniles (2 months posthatch) were obtained from Florida Marine Aquaculture Inc. Fish were randomly combined in nine 10-L aquaria, and fed Zeigler adult zebrafish diet (Adult Zebrafish Diet by Zeigler, Aquatic Habitats) twice per day for 2 weeks. Before the experiment, fish were fed the A0B0 diet for 10 days, thrice per day, to accustom the fish to the purified diet. All the zebrafish were re-pooled, and 45 fish of both sexes were randomly distributed in three replicate 10-L plastic aquariums for each of the five dietary treatments. Each treatment was applied in triplicate following a completely randomized experimental design, for a total of 15 aquaria. The initial mean body mass of the fish was 0.266 g. Before the onset of the trial, 30 randomly collected individuals from the base population were killed, weighed, and stored at −20°C for initial proximate analysis according to standard procedures⁴⁰: DM was determined after drying in an oven at 105°C for 24 h; ash was determined by incineration at 550°C for 12 h; crude protein was determined by kjeldhal method (N%×6.25); and crude fat was determined by soxtec extraction with diethyl ether (40°C–60°C). Ten fish specimens were also collected and individually analyzed for initial whole-body biotin analysis according to the procedure developed by Mock and colleagues⁴¹⁻⁴⁴ as described by Staggs et al.⁴⁵ Briefly, the fish were first homogenized with teflon homogenizer, followed by acid hydrolysis to release protein-bound biotin. Biotin was separated from inactive metabolites by HPLC by using a C18 reversed-phase column. The HPLC eluate fractions were dried to remove solvents and rehydrated in assay buffer. Biotin was quantified by competitive ELISA.^42–46 DM, ash, crude lipid, crude protein, energy, whole-body biotin content, and condition factor of the initial fish were 28.50%, 3.52%, 28.89% DM, 55.34% DM, 5.97 kcal g⁻¹, 109.58 ng g⁻¹, and 1.43 respectively.

System water was treated by using a recirculating aquaculture system (RAS) (100% water replacement per day and 1.2 L min⁻¹ in each aquarium) at the Regional Aquatic Sciences Laboratory, Université Laval, over a period of 8 weeks. The system was supplied with dechlorinated water. The system contained a drum filter, a sump, and a fluidized bed biofilter of Kaldness media (Aquamerik) to maintain stable water conditions in the RAS. The photoperiod was maintained at 14:10 (light:dark) hours. The following water physico-chemical parameters were checked on a daily basis: temperature, dissolved oxygen, pH, salinity, conductivity, and hardness. The NH₃/NH₄⁺, nitrite NO₂⁻, and nitrate NO₃⁻ were checked on a weekly basis. At the end of the study, water samples were collected from the RAS for biotin measurement in water, by competitive ELISA.^41–44 The water quality parameters remained stable and within acceptable ranges^8,47 during the experiment. Water temperature ranged from 25.9°C to 26.6°C, dissolved oxygen from 6.9 to 7.9 mg L⁻¹, total NH₃ from 0.0000 to 0.0009 mg L⁻¹, NO⁻₂ from 0.0066 to 0.0924, NO⁻₃ from 2.2 to 6.3 mg L⁻¹, alkalinity from 2.9 to 31 mg L⁻¹ of CaCO3, salinity from 0.2 to 1.1 ppt, hardness from 67 to 114.4 mg L⁻¹ of CaCO₃, conductivity from 227 to 269.0 μS cm⁻¹, and the pH from 6.9 to 7.8. Biotin was below detection limit (<80 fmol mL⁻¹) in the water of the RAS.

During the experimental period, fish were hand-fed thrice per day (08:00, 13:00, and 17:00) to apparent satiety. Uneaten food particles were collected, and their DM content was determined. Mortality was recorded every day during the trial. The body of each dead fish was examined for signs of biotin deficiency. The study lasted 8 weeks. The fish were not fed the afternoon before the final sampling day. At the end of the experiment, fish from each aquarium were anaesthetized with clove oil/eugenol (60 mg L⁻¹),⁴⁸ bulk weighed, and individually counted; and feed conversion ratio (FCR), growth parameters, protein efficiency ratio (PER), energy conversion efficiency (ECE), and survival rate were calculated.

Three fish per aquarium were randomly sampled, euthanized by an overdose of clove oil/eugenol (150–200 mg L⁻¹),⁴⁸ their individual weight after blotting excess water with a paper towel and standard length were measured, and they were analyzed for whole-body biotin content as previously described.^41–44,46 The remaining fish were euthanized by an overdose of clove oil/eugenol (150–200 mg L⁻¹),⁴⁸ then individually weighed, their fork length measured for growth parameters, condition factor, and food conversion parameters, and they were finally used for final proximate composition analysis.⁴⁰

Statistical methods

Each aquarium was considered an experimental unit. Results were expressed as mean (n=3). Significance of differences between group means was analyzed by one-way ANOVA and was deemed significant if p<0.05. When significant differences were observed, then multiple comparisons among means were made with Duncan's multiple range test by using SAS version 9.2.

Results

Growth performance, survival rate, feed utilization, and condition factor

The effects of the diets were significant on growth, survival rate, feed utilization, and condition factor of zebrafish (p<0.05) (Table 2). The percentage of weight gain was similar between the groups of fish fed the ZC and A0B1 diets, and between the groups fed the A120B0 and A0B0diets, with the first groups showing the higher values; the lowest percentage weigh gain was obtained with the diet supplemented with antibiotic (A0B0S). The results of the final weight and specific growth rate followed the same pattern, with the exception that the ZC diet showed higher values than the A0B1 diet. The lowest survival rate was obtained with the diet supplemented with antibiotic (A0B0S), whereas the highest value was obtained with the practical ZC diet and the diet containing the biotin requirement level for zebrafish (A0B1), followed by the diets A0B0 and A120B0 respectively. The PER and protein conversion efficiency were similar between the groups of fish fed the ZC and A0B1 diets, and the lowest values were obtained with the diet supplemented with antibiotic (A0B0S). The FCR was similar between the groups of fish fed the ZC and A0B1 diets, which showed the best (lowest) values. The highest FCR was obtained with the diet supplemented with antibiotic (A0B0S), followed by the diet A120B0 and A0B0S, respectively. Zebrafish condition was similar in the diet ZC and the purified diet containing the biotin requirement level for zebrafish (A0B1), which was higher than that observed in the nonbiotin and nonavidin supplemented diet (A0B0); the lowest value was obtained with the diets A120B0 and A0B0S.

Table 2.

Final Weight, Percentage Weight Gain, Specific Growth Rate, Protein Efficiency Ratio, Protein Conversion Efficiency, Energy Conversion Efficiency, Survival Rate, Feed Conversion Ratio, and Condition Factor of Zebrafish Danio rerio Fed Diets with Various Biotin Statuses for 8 Weeks ¹

	Experimental diets ²
Parameters	ZC diet	A0B0	A0B1	A120B0	A0B0S	p-value	Pooled SEM
Initial weight (g)	0.266	0.265	0.267	0.266	0.265
Final weight (g)	0.618^a	0.520^c	0.594^b	0.507^c	0.449^d	<0.0001	0.007
Percentage weight gain (%)³	128.15^a	96.25^b	122.21^a	90.56^b	69.06^c	<0.0001	2.161
SGR (%day⁻¹)⁴	1.50^a	1.20^c	1.43^b	1.15^c	0.94^d	<0.0001	0.024
Survival rate (%)⁵	95.56^a	90.37^b	94.82^ab	77.04^c	51.85^d	<0.0001	3.444
PER (g g⁻¹)⁶	1.031^a	0.769^b	1.000^a	0.687^c	0.503^d	<0.0001	0.018
PCE (g g⁻¹)⁷	0.180^a	0.122^b	0.166^a	0.111^b	0.030^c	<0.0001	0.006
ECE (kcal g⁻¹)⁸	0.207^a	0.100^c	0.139^b	0.103^c	0.049^d	<0.0001	0.004
FCR⁹	1.685^d	2.211^c	1.723^d	2.473^b	3.340^a	<0.0001	0.049
Condition factor¹⁰	1.51^a	1.15^b	1.48^a	1.05^c	1.06^c	<0.0001	0.010

Means with different superscript letters in a row are significantly different (p<0.05).

Average Weight Gain (%)=((final wet weight (g)−initial wet weight (g))/initial wet weight (g))^*100.

SGR (%/day)=(ln final wet weight (g)–ln initial wet weight (g))/Time (days).

Survival rate (%)=(Final number of fish Nf/Initial number of fish)×100.

Protein efficiency rate PER (g/g)=(final wet weight (g) − initial wet weight (g))/(Dry feed consumed (g)×Protein content of the feed (% DM)).

PCE=[((final wet weight (g)×Final protein content of the fish (%)) − (Initial wet weight (g)×Initial protein content of the fish (%))]/(Dry feed consumed (g)×Protein content of the feed (% DM)).

ECE=[((final wet weight (g)×Final energy content of the fish (%)) − (Initial wet weight (g)×Initial energy content of the fish (%))]/(Dry feed consumed (g)×Energy content of the feed (% DM)).

FCR (Food conversion ratio) (g/g)=(Quantity of feed distributed (g)×Dry matter content of feed)/(final wet weight (g) − initial wet weight (g)).

Condition factor: 10⁵×mass (g)×fork length (mm)⁻³.

FCR, feed conversion ratio; PER, protein efficiency ratio; ECE, energy conversion efficiency; SGR, specific growth rate; PCE, protein conversion efficiency.

Zebrafish proximate composition and whole-body biotin

The effects of the diets were significant in the proximate composition (except on ash content) and whole-body biotin content of zebrafish (p<0.05) (Table 3). Fish fed the practical ZC diet showed the higher proximate compositions than those observed in fish fed the purified diets, whereas the lowest values were observed in fish fed the diet supplemented with antibiotic (A0B0S). The whole-body biotin content of the fish was higher in ZC diet, followed by the diet supplemented with 120×more avidin than the requirement biotin level (A120B0), and the lowest value was obtained in fish fed the A0B0S diet.

Table 3.

Body Proximate Composition and Whole-Body Biotin Content of Zebrafish Danio rerio Fed Diets with Various Biotin Statuses for 8 Weeks ¹

	Experimental diets ²
Parameters	ZC diet	A0B0	A0B1	A120B0	A0B0S	p-value	Pooled SEM
DM (%)	30.07^a	26.06^c	27.01^b	27.39^b	25.78^c	<0.0001	0.255
Ash (%)	3.45	3.85	3.95	3.98	3.96	0.3015	0.189
Crude protein (% DM)	29.19^a	21.16^b	21.98^b	21.29^b	19.26^c	<0.0001	0.716
Crude lipid (% DM)	55.61^c	60.83^a	60.09^ab	58.16^b	45.85^d	<0.0001	0.302
Gross energy (kcal g⁻¹)	6.04^a	5.60^b	5.57^b	5.69^b	5.43^c	<0.0001	0.045
Biotin content (ng g⁻¹)	385.52^a	68.96^d	96.52^c	347.86^b	24.22^e	<0.0001	6.664

Means with different superscript letters within a row are significantly different (p<0.05).

A0B0, A0B1, and A120B0 refer to the basal purified diets where the molar avidin:biotin ratio's added are 0:0, 0:1, and 120:0, respectively, whereas A0B0S refers to the basal diet containing the antibiotic succinylsulfathiazole (1%, wt/wt) with neither biotin nor avidin supplementation; ZC diet refers to Zeigler commercial diet for zebrafish (Adult Zebrafish Diet by Zeigler, Aquatic Habitats).

Discussion

The current study aimed at preliminarily assessing the contribution of the intestinal microflora to biotin supply for growth and survival of zebrafish, by suppressing this microflora with a supplementation of the antibiotic succinylsulfathiazole (1%, wt/wt) in the diet. Succinylsulfathiazole has been extensively used in previous studies to assess the contribution of the intestinal microflora to vitamin supply in both juvenile and adult fish, and growth performance, tissue-vitamin concentration, survival, and feed efficiency were reduced in fish species that required vitamin supply from this intestinal microflora.^35–39

The results of the current study suggested that the intestinal microbial synthesis is a significant source of biotin to the host, the zebrafish. This contribution of the intestinal microflora is revealed by the reduction in growth, condition factor, whole-body biotin content, survival, and an increase in FCR observed in the fish fed the diet containing the antibiotic with neither biotin nor avidin supplementation (A0B0S), compared with the diet A0B0 and A0B1. When comparing the fish growth and survival obtained with the diets A0B0 and A0B0S, it appears that the intestinal microflora of zebrafish might relatively contribute to increase the growth by 15.8%, survival by 74.3%, whole-body biotin content by 184.7%, and the condition factor by 8.5%, and to reduce the FCR by 33.8%. Further, in the specific case of zebrafish, the effects of the antibiotic in biotin-deficiency induction seems higher than that of dietary avidin, as growth, survival, and feed utilization are lower with the diet supplemented with antibiotic than that supplemented with avidin (A120B0). Therefore, it is likely that the supplementation of succinylsulfathiazole in the diet effectively suppressed intestinal synthesis of biotin in zebrafish. This assumption is in line with the results of Stokstad,⁴⁹ who noted severe biotin deficiency signs on feeding rats a purified diet supplemented with succinylsulfathiazole. In general, vitamins have been found to be synthesized by intestinal microflora and absorbed at the colon in freshwater fishes,³³ as was reported for vitamin B-12^35,50 and biotin²⁷ in channel catfish, as well as for vitamin B-12, niacin, panthothenic acid, thiamine, and riboflavin in carp.⁵¹ The production of the biotin by the intestinal microflora and its absorption by the fish has been reported for ayu fish Plecoglossus altivelis,³³ which harbors biotin-producing bacteria as a major microbial community and biotin-consuming bacteria as a minor microbial community in the intestine. However, the mechanism or site of absorption of intestinally synthesized vitamins by fish is unknown.²⁷ However, it has been found that significant absorption of biotin from the proximal colon occurs, which gives credence to the concept that biotin from microbial synthesis within the colon can contribute to meeting the requirement for humans⁵² and may make a significant contribution to biotin nutrition in animals in general.⁵³ Further studies on identification and quantification of biotin-producing bacteria in the gut of zebrafish, and the absorption of such endogenously produced biotin, would be informative. The other possible negative effects of succinylsulfathiazole on fish health has not yet been described, but the similarity in fish condition between the groups of fish fed the antibiotic supplemented diet and the avidin supplemented diet suggests that both diets had the same general adverse effects on fish health, which were reflected by severe biotin deficiency signs.

On the other hand, although feeding the commercial diet led to the highest final weight, ECE as well as the whole-body biotin content of zebrafish in the current study, there is a similarity in terms of condition factor, PER, FCR, percentage weight gain, and survival rate of zebrafish between the fish groups fed the commercial diet (ZC) and the biotin supplemented diet (A0B1). These results supported our previous conclusions that 0.51 mg biotin kg⁻¹ diet appear to meet the biotin requirement of zebrafish,^11,24 and the performance obtained with this purified diet is comparable to that obtained with the commercial diet. Using such a purified diet has the advantage of controlling the ingredients and nutrients that are used in the formulation, and, therefore, considerably limits the confounding research results caused by nutritional vagaries that can happen with the practical (commercial) diet.⁴ The results of the present experiment also confirmed the essentiality of dietary biotin for zebrafish,^11,24 as the growth and survival of the fish was reduced, and the FCR was increased when fish were fed the diet free of both biotin and avidin (A0B0) compared with the diet supplemented with the biotin requirement level for zebrafish (A0B1) that was determined in a previous study by Yossa et al.¹¹ Dietary biotin has been found to be required for maximal growth of many other fish species, among which the following are common: carp Cyprinus carpio,²⁵ hybrid tilapia Oreochromis niliticus x O. aureus,³⁰ lake trout Salvelinus namaycus,⁵⁴ rainbow trout Oncorhynchus mykiss,²⁶ Atlantic salmon Salmo salar,¹³ channel catfish Ictalurus punctatus,^27,23 mirror carp,²⁸ Indian catfish Clarias batrachus,⁹ and, more recently, Japanese seabass Lateolabrax japonicus.⁵⁵

In the current study, the supplementation of avidin in zebrafish diet (A120B0) led to the same growth, but lower survival and lower condition factor, as well as higher FCR than that observed in fish fed the nonbiotin and nonavidin supplemented diet (A0B0). It has been previously reported that avidin antagonizes biotin by forming with the vitamin a non-covalent complex that is also resistant to pancreatic proteases, thus preventing the absorption of biotin in the gastrointestinal tract and inducing biotin-deficiency signs.^10,21,56 The same biotin deficiency signs observed in the current study, due to impairing effects of the dietary avidin on growth, survival, and feed utilization of zebrafish, were noted in a previous study where gradual levels of dietary avidin were supplemented to zebrafish diets.²⁴ These findings are in accordance with the growth reductions found in biotin-deficient rainbow trout,²⁶ Atlantic salmon fry,¹³ Indian catfish,³¹ and hybrid tilapia.³⁰ Several investigators reported high mortality of Atlantic salmon and channel catfish, respectively, after feeding diets supplemented with dried egg white used as avidin source^13,23 and also reduced feed utilization.²³

Conclusion

Intestinal microbial synthesis is assumed to be a significant source of biotin to the zebrafish, as fish fed the antibiotic-supplemented diet showed the lowest growth, health condition, and feed utilization. Further studies on identification and quantification of biotin-producing microflora in the gut of zebrafish, and possibly the absorption of such endogenously produced biotin, would be informative.

Footnotes

Acknowledgments

This study was funded by the National Sciences and Engineering Research Council of Canada. The authors would like to thank Émilie Proulx, Micheline Gingras, Noémie Stewart-Poirrier, and Jean-Gabriel Soulières-Jasmin at the department of Animal Sciences, Université Laval, Canada, for their kind collaboration during feeding and the biotin analysis in this study. They would also like to express their profound gratitude to the staff of the Regional Laboratory of Aquatic Sciences (LARSA) of the Université Laval, Canada, for their precious assistance in rearing the zebrafish used in the current study. The technical assistance of Dr. Donald M. Mock and Nell I Matthews, Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, in developing the biotin analysis used in the current study is greatly appreciated.

Disclosure Statement

No competing financial interests exist.

References

Fishman

. Genomics: Zebrafish, the canonical vertebrate. Science, 2001; 294:1290–1291.

Carvalho

, Araujo

, Santos

. Rearing zebrafish (Danio rerio) larvae without live food: evaluation of a commercial, a practical and a purified starter diet on larval performance. Aquac Res, 2006; 37:1107–1111.

Lawrence

. The husbandry of zebrafish (Danio rerio): a review. Aquaculture, 2007; 269:1–20.

Siccardi

III , Garris

, Jones

, Moseley

, D'Abramo

, Watts

. Growth and survival of zebrafish (Danio rerio) fed different commercial and laboratory diets. Zebrafish, 2009; 6:1–6.

Jaya-Ram

, Kuah

M-K

, Lim

P-S

, Kolkovski

, Shu-Chien

. Influence of dietary HUFA levels on reproductive performance, tissue fatty acid profile and desaturase and elongase mRNAs expression in female zebrafish Danio rerio. Aquaculture, 2008; 277:275–281.

Sumanasa

, Lin

. Zebrafish as a model system for drug target screening and validation. Drug Discov. Today Targets, 2004; 3:89–96.

Markovich

, Rizzuto

, Brown

. Diet affects spawning in zebrafish. Zebrafish, 2007; 4:69–74.

Matthews

, Trevarrow

, Matthews

. A virtual tour of the guide for Zebrafish users. Lab Anim, 2002; 31:34–40.

Shaik Mohamed

, Ravisankar

, Ibrahim

. Quantifying the dietary biotin requirement of the catfish, Clarias batrachus. Aquac Int, 2000; 8:9–18.

10.

Halver

. The vitamins. Halver

, Hardy

. Fish Nutrition, 3rd. San Diego, CA: Academic Press, 2002; 61–141.

11.

Yossa

, Sarker

, Mock

, Vandenberg

. Study of the dietary biotin requirement of zebrafish Danio rerio (Hamilton-Buchanan) J Fish Physiol BiochemID no. FISH-S-11-00171.

12.

Dakshinamurti

. Biotin. Shils

, Olson

, Shike

. Modern Nutrition in Health and Disease, 8th 1. Philadelphia, PA: Lea and Febiger, 1994; 426–431.

13.

Maeland

, Waagbo

, Sandnes

, Hjeltnes

. Biotin in practical fish-meal based diet for Atlantic salmon Salmo solar L. fry. Aquac Nutr, 1998; 4:241–247.

14.

Shiau

S-Y

, Chin

Y-H

. Estimation of the dietary biotin requirement of juvenile hybrid tilapia, Oreochromis niloticus x O. aureus. Aquaculture, 1999; 170:71–78.

15.

Rodriguez-Melendez

, Zempleni

. Regulation of gene expression by biotin (Review) J Nutr Biochem, 2003; 14:680–690.

16.

Ramaswamy

. Intestinal absorption of water-soluble vitamins focus on “molecular mechanism of the intestinal biotin transport process.” Am J Physiol Cell Physiol, 1999; 277:603–604.

17.

Dakshinamurti

. Regulation of gene expression by biotin, vitamin B6 and vitamin C. Daniel

, Zempleni

. Molecular Nutrition. Oxfordshire, UK: CABI Publishing, 2003; 151–165.

18.

Dakshinamurti

. Biotin-a regulator of gene expression. J Nutr Biochem, 2005; 16:419–423.

19.

Boas

. The effect of desiccation upon the nutritive properties of egg-white. Biochem J, 1927; 21:712–724.

20.

Kuramitz

, Matsuda

, Sugawara

, Tanaka

. Electrochemical evaluation of the interaction between avidin and biotin at biotinylated polypyrrole electrode using a redox marker. Electroanalysis, 2003; 15:225–229.

21.

Combs

Jr.

The Vitamins, Fundamentals Aspects in Nutrition and Health, 3rd. San Diego, CA: Academic Press, 2008; 583.

22.

Castledine

, Cho

, Slinger

, Hicks

, Bayley

. Influence of dietary biotin level on growth, metabolism and pathology of rainbow trout. J Nutr, 1978; 108:698–711.

23.

Lovell

, Buston

. Biotin supplementation of practical diets for channel catfish. J Nutr, 1984; 114:1092–1096.

24.

Yossa

, Sarker

, Karanth

, Ekker

, Vandenberg

. Effects of dietary biotin and avidin on growth, survival, feed conversion, biotin status and gene expression of zebrafish Danio rerio (Hamilton-Buchanan) Comp Physiol Biochem Part B, 2011, 10.1016/j.cbpb.2011.07.005.

25.

Ogino

, Watanabe

, Kakino

, Iwanaga

, Mizuno

. B-vitamin requirements of carp: requirement of biotin. Bull Jpn Soc Sci Fish, 1970; 36:734–740.

26.

Woodward

, Frigg

. Dietary biotin requirements of young rainbow trout (Salmo gairdneri) determined by weight gain, hepatic biotin concentration and maximal biotin-dependent enzyme activities in liver and white muscle. J Nutr, 1989; 119:54–60.

27.

Robinson

, Lovell

. Essentiality of biotin of channel catfish, Ictalurus punctatus, fed lipid and lipid-free diets. J Nutr, 1978; 108:1600–1605.

28.

Gunther

, Meyer-Burgdorff

. Studies on biotin supply to mirror carp (Cyprinus carpio L.) Aquaculture, 1990; 84:49–60.

29.

Shiau

S-Y

, Chin

Y-H

. Dietary biotin requirement for maximum growth of juvenile grass shrimp, Penaeus monodon. J Nutr, 1998; 128:2494–2497.

30.

Shiau

S-Y

, Chin

Y-H

. Estimation of the dietary biotin requirement of juvenile hybrid tilapia, Oreochromis niloticus x O. aureus. Aquaculture, 1999; 170:71–78.

31.

Shaik Mohamed

. Dietary biotin requirement determined for Indian catfish, Heteropneustes fossilis (Bloch) fingerlings. Aquac Res, 2001; 32:709–716.

32.

Halver

. The vitamins. Halver

. Fish Nutrition, 2nd. New York: Academic Press, 1989; 57–60.

33.

Sugita

, Takahashi

, Deguchi

. Production and consumption of biotin by intestinal microflora of cultured freshwater fishes. Biosci Biotechnol Biochem, 1992; 56:1678–1679.

34.

Couch

, Cravens

, Elvehjbmand

, Halpin

. Relation of carbohydrate to intestinal synthesis of biotin and hatchability in the mature fowl. J Nutr, 1948; 35:57–72.

35.

Limsuwan

, Lovell

. Intestinal synthesis and absorption of vitamin B-12 in channel catfish. J Nutr, 1981; 111:2125–2132.

36.

Burtle

, Lovell

. Lack of response of channel catfish (Ictalurus punctatus) to dietary myo-inositol. Can J Fish Aquat Sci, 1989; 46:218–222.

37.

Duncan

, Lovell

, Butterworth

Jr. , Freeberg

, Tamura

. Dietary folate requirement determined for channel catfish, Ictalurus punctatus. J Nutr, 1993; 123:1888–1897.

38.

Deng

, Hemre

, Wilson

. Juvenile sunshine bass (Morone chrysops♀ x Morone saxatilish♂) do not require dietary myo-inositol. Aquaculture, 2002; 213:387–393.

39.

Shiau

S-Y

, Su

S-L

. Juvenile tilapia (Oreochromis niloticus x O. aureus) requires dietary myo-inositol for maximal growth. Aquaculture, 2005; 243:273–277.

40.

AOAC International. Official Methods of Analysis of AOAC International 18th. Arlington, VA, 2007.

41.

Mock

. A sequential, solid phase assay for biotin based on 125I-avidin. Methods Enzymol, 1990; 184:224–233.

42.

Mock

, Mock

, Langbehn

. Biotin in human milk: methods, location, and chemical form. J Nutr, 1992; 122:535–545.

43.

Mock

, Lankford

, Mock

. Biotin accounts for only half of the total avidin-binding substances in human serum. J Nutr, 1995; 125:941–946.

44.

Mock

, Mock

, Stratton

. Concentrations of biotin metabolites in human milk. J Pediatr, 1997; 131:456–458.

45.

Staggs

, Sealey

, McCabe

, Teague

, Mock

. Determination of the biotin content of select foods using accurate and sensitive HPLC/avidin binding. J Food Compos Anal, 2004; 17:767–776.

46.

Mock

, Malik

. Distribution of biotin in human plasma: most of the biotin is not bound to protein. Am J Clin Nutr, 1992; 56:427–432.

47.

Westerfield

. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio), 5th. Eugene, OR: University of Oregon Press, 2007.

48.

Grush

, Noakes

DLG

, Moccia

. The efficacy of clove oil as an anesthetic for the Zebrafish, Danio rerio (Hamilton) Zebrafish, 2004; 1:46–53.

49.

Stokstad

ELR

. Antibiotics in animal nutrition. Physiol Rev, 1954; 34:25–51.

50.

Sugita

, Miyajima

, Deguchi

. The vitamin B12-producing ability of the intestinal microflora of cultured freshwater fish. Aquaculture, 1991; 92:267–276.

51.

Kashiwada

, Teshima

. Studies on the production of B vitamin by intestinal bacteria of fish, I. Nicotinic acid, pantothenic acid and B12 in carp. Bull Jap Soc Sci Fish, 1966; 32:961–966.

52.

Institute of Medicine. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington, DC: National Academy Press, 2000; 564.

53.

Bender

. Nutritional Biochemistry of the Vitamins, 2nd. Cambridge, UK: Cambridge University Press, 2003; 488.

54.

Poston

. Optimum level of dietary biotin for growth, feed utilization and swimming stamina of fingerling lake trout (Salvelinus namaycush) J Fish Res Board Can, 1976; 33:1803–1806.

55.

, Zhang

, Mai

, Ai

, Wan

, Zhang

. Estimation of dietary biotin requirement of Japanese seabass, Lateolabrax japonicus C. Aquac Nutr, 2010; 16:231–236.

56.

De Silva

, Anderson

. Fish Nutrition in Aquaculture. London: Chapman and Hall Aquaculture Series, 1995; 319.