Growth Performance,Body Lipid,Brood Amount,and Rearing Environment Response to Supplemental Neutral Phytase in Zebrafish ( Danio Rerio ) Diet

Abstract

The present study was to evaluate the effects of neutral phytase supplementation on growth performance, survival ratio (SR), body lipid, brood amount, and rearing environment in zebrafish. The control diet was not supplemented phytase, and three levels of phytase (500, 1000, or 1500 U kg⁻¹) was added to the three other diets (named as PP500, PP1000, and PP1500). Triplicate groups (twelve 100-L tanks) of zebrafish (initial mean weight, 0.284±0.012 g) were fed twice daily (08:00 and 16:00 h) to satiation for 12 weeks. The results showed that supplemental phytase in the diet improved weight gain (60.49%, 86.63%, 99.06%, and 111.88% in control, PP500, PP1000, and PP1500) and the specific growth ratio of zebrafish (p<0.05). Dietary phytase addition increased the whole body lipid content of zebrafish. The brood amounts (116, 123, and 124 eggs in PP500, PP1000, and PP1500) of fish fed with phytase-supplemented diets were little higher than the control (mean egg was 112). The ammonia–nitrogen concentration in water of fish fed with phytase-supplemented diet was significantly lower than the control. The nitrite concentration in water was also decreased in water of fish fed with phytase-supplemented diet. The SR was increased with the increasing of dietary phytase despite no significant difference was observed among each group. The present study first suggested that neutral phytase could be applied in the zebrafish diet. Furthermore, phytase addition increased the growth, body lipid, brood amount, and SR of zebrafish, and meanwhile decreased the ammonia–nitrogen and nitrite concentrations in rearing water.

Introduction

The need to develop standardized diets to support zebrafish (Danio rerio) research is supported by the fact that specific dietary ingredients, nutrients, or antinutritional factors (ANFs) in diets have been shown to affect development and growth of adult D. rerio and their offspring.¹ One of the main ANFs is phytate, which is the primary storage form of mineral in seeds. It hinders the digestibility of protein and minerals in rainbow trout (Salmo gairdneri)^2,3 causing depressed growth of rainbow trout (S. gairdneri)¹ and juvenile Chinook salmon (Oncorhynchus tshawytscha).⁴ The availability of minerals and other nutrients can be significantly enhanced in grass carp (Ctenopharyngodon idellus) by the addition of phytase.⁵ Studies on the role of supplemental phytase for growth or nutrient utilization in fish have principally focused on commonly cultured species such as Atlantic salmon (Salmo salar L.),⁶ rainbow trout (Oncorhynchus mykiss Walbaum),⁷ Nile tilapia (Oreochromis niloticus),⁸ gibel carp Carassius auratus gibelio,⁹ and common carp (Cyprinus carpio L.).¹⁰

Recently, zebrafish have emerged as a model organism because of the biological advantages that include their small size, short generation time interval, their capacity to produce numerous offspring, breed easily, and very amenable to manipulation in a laboratory tank.¹¹ It has also been widely used in neurobiology and molecular genetics.¹² Zebrafish have even been proposed as a possible model organism for nutrition and growth studies in fish.^13,14 Many variations have occurred in zebrafish growth rates in laboratories,¹⁵ but uniform growth and high survivability are difficult to ensure. Other aspects such as reproduction and aquaculture environment were largely studied,¹⁶ but little attention was given to the effect of the nutrition factor. According to the information on the dietary preferences, zebrafish can be maintained and successfully spawned under a wide variety of diets and feeding regimes,¹⁷ which allows us to infer that it has nutritional pathways similar to those of cultivated herbivores such as carp and tilapia. Previous study indicated that phytase increased the growth of fish,^6–10 however, the study of the effects of specific dietary ANFs, such as phytate, on growth of zebrafish is limited, still less phytase application in the diet of zebrafish until now.

Although zebrafish has been put forward as an ideal model organism for studying fish nutrition utilization and growth performance, only limited orienting information is available so far.¹⁷ Therefore, the present study aims at evaluating the effects of neutral phytase supplementation on growth performance, body lipid, and brood amount of zebrafish, as well as rearing water quality. This study will be helpful in confirming whether neutral phytase could be applied in the zebrafish diet or not.

Materials and Methods

Experimental diets

Three isonitrogenous and isocaloric experimental diets are formulated as no phytase supplementation (control), phytase pretreatment with 500 U (PP500), 1000 U (PP1000), or 1500 U (PP1500) per kg diet. The ingredients and analyzed compositions of four diets are presented in Table 1. The diets were made into pellets of 2 mm diameter by a laboratory pellet machine and broken into small pellets by a crusher. The neutral phytase with an optimal pH about 7.0 well adapt to the intestinal pH of carp¹⁸ and may be a good candidate for an aquatic feed additive in the aquaculture industry. Neutral phytase was isolated from Pedobacter sp. MJ11 (CGMCC No. 2503) and designated as PHYMJ11, was synthesized by SunHY Biology Co., Ltd. (Wuhan, China) with enzyme activity of 2500 U g⁻¹.

Table 1.

Ingredient and Formula Analysis of the Experimental Diets

Item	Control	P500	P1000	P1500
Ingredients^a (g kg⁻¹ diet)
Soybean meal	500	500	500	500
Fish meal^b	160	160	160	160
Casein	150	150	150	150
Fish oil	10	10	10	10
Soybean oil	10	10	10	10
Corn starch	120	119.8	119.6	119.4
Vitamin premix^c	10	10	10	10
Mineral premix^d	10	10	10	10
Monocalcium phosphate	20	20	20	20
Carboxymethylcellulose sodium	10	10	10	10
Neutral phytase^e	0	0.2	0.4	0.6
Compositions (%)
Crude protein	46.28	46.66	46.44	46.71
Crude lipid	5.45	5.17	5.38	5.15
Ash	6.48	6.22	6.51	6.34
Moisture	9.54	9.18	9.31	9.47
Phosphorus	1.14	1.12	1.16	1.09
Calcium	0.57	0.61	0.59	0.64

Ingredients, Fu Long Dietary Company, Wuhan, China.

Fish meal, Peru fishmeal.

Vitamin premix (per kg of diet): vitamin A, 2000 IU; vitamin B₁ (thiamin), 5 mg; vitamin B₂ (riboflavin), 5 mg; vitamin B₆, 5 mg; vitamin B₁₂, 0.025 mg; vitamin D₃, 1200 IU; vitamin E, 21 mg; vitamin K₃, 2.5 mg; folic acid, 1.3 mg; biotin, 0.05 mg; pantothenic acid calcium, 20 mg; inositol, 60 mg; ascorbic acid (35%), 110 mg; niacinamide, 25 mg.

Mineral premix (per kg of diet): MnSO₄, 10 mg; MgSO₄, 10 mg; KCl, 95 mg; NaCl, 165 mg; ZnSO₄, 20 mg; KI, 1 mg; CuSO₄, 12.5 mg; FeSO₄, 105 mg; Na₂SeO₃, 0.1 mg; Co, 1.5 mg.

Neutral phytase, the activity was 2500 U g⁻¹.

Fish and experimental conditions

About 800 zebrafish were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China. Before the experiment, fish were reared in four 300-L round tanks and fed with a control diet for 2 weeks to adjust to the experimental diet. After conditioning for 2 weeks, fish were starved for 24 h to measure the body length and weight at the beginning of experiment. Then, the fish were randomly distributed among twelve 100-L tanks (with 80-L water), where the fish were acclimated to the new rearing conditions. Stocking densities were 40 fish (mean weight, 0.284±0.012 g) per tank and each treatment being randomly assigned to triplicate tanks of fish. Dissolved oxygen was 7.22±0.43 mg L⁻¹, temperature ranged from 23°C to 29°C, and pH was about 6.86±0.29. During the trial period, fish were fed to apparent satiation with the experimental diets twice per day at 08:30 and 16:00 for 12 weeks. The residual feed was settled at the end of feeding by Guelph-type settlement collectors 30 min later.

Sample collection and analyses

At the end of the 12-week trial, approximately 24 h after the last feeding, all fish were weighed to determine the weight gain (WG) and specific growth ratio (SGR). After that, the feed conversion ratio (FCR) and protein efficiency ratio (PER) were calculated. After obtaining the final weight of all fish, twenty fish from each tank were randomly selected, dried at 60°C for subsequent determination of the whole body proximate composition. Proximate analysis, including moisture, crude protein, and crude lipid contents were done following standard methods.¹⁹ Moisture was determined by oven drying at 105°C for 6 h. Crude protein (N×6.25) of the samples was determined by the Kjeldahl method after an acid digestion using a Kjeltec system (Kjeltec 2300 Analyzer; Foss Tecator). Crude lipid was assayed by the ether extraction method using Soxtec System HT (Soxtec System HT6; Tecator). For checking the ammonia–nitrogen and nitrite concentrations in water, we collected water samples during each weekend, for 12 weeks. After collecting the water samples, the old water in tanks would be changed by new water each week. The measuring method of the concentrations of ammonia–nitrogen and nitrite was referenced by Mulder et al.²⁰ At the last week of the present experiment, the brood amount in mature female zebrafish was counted according to Ankley et al.²¹ We chose three fish from each tank for counting their brood amount and repeated two times (six samples in each tank).

Calculation and chemical analyses

The data obtained were analyzed for WG, SGR, FCR, PER, and the survival ratio (SR) using the following formulae:

WG (%)=100×(final weight−initial weight)/initial weight;

SGR (%/d)=100×(ln final weight−ln initial weight)/days;

FCR=total dry feed intake (g)/wet WG (g);

PER=wet WG (g)/protein fed (g);

SR (%)=100×(final fish number)/(initial fish number).

Statistical analyses

All statistical analyses were carried out using the SPSS program for Windows (v. 11.5). Data are expressed as the mean±standard error (SE) of three replicates. The means within each treatment and among treatments were subjected to one-way ANOVA and compared using the Duncan's multiple range test. The differences were considered statistically significant at p<0.05.

Results

The results showed that supplemental phytase in the diet significantly (p<0.05) improved WG and SGR of zebrafish (showed in Table 2). Fish fed the diet P1500 showed the highest WG, while fish fed the control diet showed the lowest WG. The FI was increased significantly in fish fed with phytase-supplemented diets and the highest FI was observed in fish fed the diet P1500. However, the PER was significantly elevated in fish fed with phytase-supplemented diets. The FCR was decreased in fish fed with phytase-supplemented diets compared with the control, especially in fish fed with P1000 and P1500 (p<0.05). The SR was increased with the increasing of dietary phytase although there was no significant difference from each group.

Table 2.

Growth Performance of Zebrafish Fed with the Experimental Diets for 12 Weeks

	Control	P500	P1000	P1500	p-Value
IW (g)	0.281±0.004	0.275±0.009	0.298±0.014	0.282±0.007	0.242
FW (g)	0.451±0.012^a	0.514±0.026^b	0.592±0.024^c	0.596±0.019^c	0.000
WG (%)	60.49±2.13^a	86.63±3.38^b	99.06±3.80^b	111.88±5.48^c	0.000
SGR (%)	0.562±0.016^a	0.743±0.022^b	0.818±0.023^bc	0.892±0.031^c	0.000
FI (g)	40.29±3.48^a	49.46±0.82^b	56.55±1.61^c	58.21±1.10^c	0.001
FCR	7.28±0.42^c	5.86±0.27^b	5.09±0.35^a	4.75±0.10^a	0.000
PER	0.300±0.017^a	0.366±0.004^b	0.417±0.013^c	0.451±0.016^d	0.000
SR (%)	93.33±3.63	95.00±2.89	97.50±0.00	98.33±0.83	0.462

Values are mean±standard error of three replicates and values within the same row with different superscript letters are significantly different (p<0.05).

IW, initial weight; FW, final weight; WG, weight gain; SGR, specific growth ratio; FI, feed intake; FCR, feed conversion ratio; PER, protein efficiency ratio; SR, survival ratio.

The body lipid content was significantly increased in fish fed with phytase-supplemented diets compared with the control (Fig. 1A), and it was increased with the increasing of dietary phytase supplementation. Conversely, the body moisture content was reduced when phytase was supplemented, and decreased significantly in fish fed with high phytase (1000 and 1500 U kg⁻¹) (Fig. 1D). However, no significant difference was observed in the body protein among all the groups (p>0.05) (Fig. 1C).

FIG. 1.

The body lipid (% dry matter) (A), brood amount (B), body protein (% dry matter) (C), and body moisture (D) of fish fed with phytase-supplemented diets and the control diet. The ammonia–nitrogen concentration (E) and nitrite concentration (F) in water of fish fed with phytase-supplemented diets and the control diet. Columns represent the mean. Bars represent the standard error (SE). Treatments that do not share a common letter are significantly different from each other as determined by one-way ANOVA (p<0.05). Values are mean±SE (n=3).

The brood amount was counted in mature female zebrafish and six samples in each tank were chosen. The mean brood amount in the control was 112 eggs. In PP500, PP1000, and PP1500, the respective mean brood amount was 116, 123, and 124 eggs (Fig. 1B). Phytase increased the amount of fish brood in spite of no significant difference observed among all the groups.

The ammonia–nitrogen concentration in water of fish fed with the phytase-supplemented diet was significantly lower than the control (Fig. 1E), and the lowest concentration was observed in water of fish fed with P1500. The nitrite concentration was also decreased in water of fish fed with the phytase supplementation diet, especially (p<0.05) in water of fish fed with P1000 and P1500 (Fig. 1F).

Discussion

The present study showed that the dietary phytase supplementation improved WG and SGR of zebrafish. The increase in WG could partially be attributed to the increased mineral bioavailability in the phytase treatment diet. The application of phytase resulted in increased ADCs of proteins and minerals.⁵ Another main reason for the increase in WG was the increase of feed intake. In the present study, decreased FCR values were conformable to the observations of Li and Robinson²² who reported enhanced feed consumption and growth performance, and lower FCR in fish fed with the phytase-supplemented diet (250 U kg⁻¹) as compared with the control. Cao et al.⁸ found that phytase pretreatment of diets increased P and other minerals in the diet, and enhanced the WG and SGR in Nile tilapia (O. niloticus). Similar results had been observed in other studies,²³ where phytase-supplemented diets increased the WG.

In the present study, the increased PER could be explained by the elevated level of crude protein apparent digestibility with phytase addition in the diets. Significant enhancement in PER because of phytase treatment has been reported in rainbow trout (O. mykiss Walbaum)⁷ and other fish species.⁸ This suggests that phytase addition or heating of meals during the treatment process improves the nutritional quality of mixed plant meals.

Supplementation of neutral phytase increased the body lipid of zebrafish in the present study. The possible reason of this phenomenon was that the dietary valuable nutrients were increased with the function of phytase. These increased available minerals might be the key element in the enzyme producing lipid metabolism. In the previous study, rainbow trout (O. mykiss) fed with phytase stored more lipids in their bodies than fish fed no phytase.²³ However, no significant difference in whole-body lipid was observed in fish fed with phytase or not.⁶ The inconsistent result among these studies was associated with different fish species, dietary ingredients, and an aquaculture environment.

Furthermore, the increasing of body lipid was an important factor in the reproduction of fish. In fish, loss of energy concomitant with gonad depletion and pre- and postreproductive behavior were associated with reductions in the body mass²⁴ and growth rate.²⁵ These changes in mass and growth could probably be attributed to the initial allocation and subsequent loss of lipids utilized for reproduction.²⁴ For evaluating the effect on reproduction, we evaluated the brood amount of zebrafish when fish were fed with graded levels of phytase. In the present study, the amount of brood was increased in fish fed with phytase despite there being no significant difference among all the groups. Thus, the supplement of phytase was beneficial for the reproduction of zebrafish. As far as we known, little reference was related with the effect of phytase on fish reproduction. The possible reason was that the usual study of phytase addition was interested in young fish for obtaining a well growth data, while the fish reproduction could not be studied.

The requirement of larval zebrafish to feed almost continuously must be balanced with the need of maintaining adequate water quality in their environment. High food inputs have a tendency to result in poor environmental conditions (low dissolved oxygen, elevated ammonia, and other factors) that will negatively impact the growth and survival of larvae. Ammonia is toxic to all vertebrates, causing convulsions, coma, and death, probably because the elevated NH₄⁺ displaces K⁺ and depolarizes neurons.²⁶ The WG of Spirodela polyrrhiza under the experimental conditions was found to decrease with increasing concentrations of total ammonia.²⁷ In the present study, the ammonia–nitrogen concentration in water of fish fed with the phytase supplementation diet was significantly lower than the control, which might be interpreted by the factors that a better growth was observed in fish fed with phytase.

The nitrite concentration was also decreased in water of fish fed with the phytase-supplemented diet in the present study. The intermediate product of this conversion, nitrite is toxic to fish, and can be problematic in freshwater systems at concentrations in excess of 1 ppm,²⁸ although small-bodied fish seem to be generally less sensitive to nitrite toxicity than larger fish.²⁹ Anecdotal evidence suggests that larval zebrafish are tolerant to nitrite at a level of up to 2.0 ppm, although these data have not been formally investigated.¹⁷ In the present study, supplemental phytase decreased the ammonia–nitrogen and nitrite concentrations in rearing water, thus phytase could be used in the zebrafish diet as an environmental friendly additive. Studies delineating tolerance levels of both developing and mature zebrafish to ammonia, nitrite, and nitrates are needed to define the standard ranges for these parameters in captivity.

Conclusions

The results of the present study indicated that supplemental neutral phytase in the diet improved WG, SGR, and PER of zebrafish. Moreover, dietary phytase addition increased the body lipid content and impacted brood amount of zebrafish. The ammonia–nitrogen and nitrite concentrations were decreased in water of fish fed with the phytase-supplemented diet. The present study first suggested that the neutral phytase could be applied in the zebrafish diet. Further studies covering various aspects of neutral phytase application are required in a model fish.

Footnotes

Acknowledgment

This work was financially supported by the National Basic Research Program of China (2009CB118702), the Special Fund for Agro-Scientific Research in the Public Interest of China (201003020), and the Huazhong Agricultural University Scientific & Technological Self-innovation Foundation (2010SC12).

Disclosure Statement

No competing financial interests exist.

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