Junwei LI, Zhencai YANG*, Xiaoling HAN, Quansen XIEand Haiyan LIU

1Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization of Ministry of Agriculture of China, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China

2College of Life Science, Hebei Normal University, Shijiazhuang 050024, Hebei Province, China

Effects of Dietary Vitamins A, B2, and B6Supplementation on Growth and Feed Utilization of Juvenile Chinese Soft-shelled Turtle Pelodiscus sinensis according to an Orthogonal Array Experiment

Junwei LI1,2, Zhencai YANG2*, Xiaoling HAN2, Quansen XIE2and Haiyan LIU2

1Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization of Ministry of Agriculture of China, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China

2College of Life Science, Hebei Normal University, Shijiazhuang 050024, Hebei Province, China

An orthogonal experimental design OA9(33) was used to evaluate the effects of vitamins (A, B2, and B6) on the growth and digestive ability of the juvenile Chinese soft-shelled turtle, Pelodiscus sinensis (initial weight, 5.9±0.2 g). A total of 135 turtles were divided into 9 groups, which each included 15 individuals. The results revealed that vitamin A (VA) had the strongest impacts on the growth rate and feed utilization among the three vitamins; 35,000 IU kg-1VA had optimal effects on the feeding intake and specifc growth rate, and 20,000 IU kg-1VA had optimal effects on protein digestibility and the feed conversion ratio. Vitamin B2(VB2) was essential for regulating protein deposition and the energy effciency for growth of the turtles; 120 mg kg-1VB2resulted in increased protein and energy deposition, and 180 mg kg-1VB2had greater benefcial effects on the growth rate. Vitamin B6(VB6) had important effects on protein and feed efficiency; however, VB6at an excessive level (120 mg kg-1) restricted turtle growth. Based on the above growth results, dietary supplementation of VA, VB2and VB6at levels of 35,000 IU kg-1, 180 mg kg-1and 70 mg kg-1, respectively, were recommended for the juvenile soft-shelled turtle.

Pelodiscus sinensis, vitamin, growth performance, digestion capacity, orthogonal design

1. Introduction

The Chinese soft-shelled turtle, Pelodiscus sinensis, is one of the most commercially important reptile species in China (Xie et al., 2012; Pu and Niu, 2013), and its total production reached 341,288 tons in 2014 (Fisheries Department of Agriculture Ministry of China, 2014). The researches on the bioenergetics and nutritional requirements of soft turtles have been reported (Nuangsaeng and Boonyaratapalin, 2001; Huang et al., 2003; Huang and Lin, 2004; Zhou et al., 2004; Hou et al., 2013; Chen and Huang, 2014); however, supplementation of the diets of these reptiles with several vitamins must be optimized for better growth performance. Vitamins play many important roles in the growth, physiology and metabolism of developing animals (Halver, 2003) and can affect the feeding and skeletal development of larval fsh (Fernández and Gisbert, 2011; Reham et al., 2013). The availability of vitamins at optimal levels is essential for normal animal growth. Previous studies have shown that vitamin A (VA) (Yutaka et al., 2011; Chen and Huang, 2014), B2(VB2) (Deng and Wilson, 2003) and B6(VB6) (Giri et al., 1997) are essential for animal growth.

Among these vitamins, VA (retinoids) includes a group of compounds that are structurally similar and exhibit biological activity due to retinol; these compounds bind to or activate a specific receptor or group of receptors (Hemre et al., 2004; Reham et al., 2013). VA is essential for maintenance of normal vision and growth in fish (Olson, 1991; Funkenstein, 2001); in addition, it enhances development of the alimentary tract (Lahov and Regelson, 1996). Previous studies have shown thatall vertebrate species can suffer from VA defciency and/ or toxicity, and the biological consequences of both deficiency and toxicity are similar among most species. Normal growth and reproduction can only be sustained in the presence of optimal VA levels (Stéphanie et al., 2010). The requirement for VA in turtles has been shown to be approximately 2.5–3.5 mg kg-1in a single factor experiment (Chen and Huang, 2014).

Riboflavin (VB2) is a water-soluble vitamin required by all animals (Deng and Wilson, 2003; Souto et al., 2008). It cannot be synthesized by monogastric animals, which must therefore consume foods with suffcient VB2levels to meet their metabolic demands (Kavita et al., 1996). A low VB2level, especially in fish, results in several signs of gross defciency, including high mortality, uncoordinated swimming, photophobia, cataracts, dark skin coloration, low feed conversion efficiency, cornea and eye lens opacity, and dark body pigmentation (NRC, 1993; Deng and Wilson, 2003); in addition, high dietary VB2intake is necessary to support maximum weight gain in fsh (Serrini et al., 1996).

VB6is the precursor of the coenzyme pyridoxal phosphate, which is required for the non-oxidative degradation of amino acids through transamination, deamination, and desulfuration. VB6metabolism is related to dietary protein or amino acid metabolism in animals (Hilton, 1989; Giri et al., 1997), and the structures and functions of digestive and immune system in fsh are affected by this vitamin (He et al., 2009; Feng et al., 2010; Li et al., 2010). Due to its multiple roles in various metabolic processes, a number of potential signs are indicative a VB6defciency in animals. In fsh, these signs include anorexia, anemia, dark coloration, loss of equilibrium, poor growth, and high mortality (Albrektsen et al., 1993; Giri et al., 1997). However, little information is available on the dietary VB2and VB6requirement of the soft-shelled turtle.

Many experiments have been conducted investigating VA, VB2and VB6requirements in aquatic animals (Halver, 1989; Serrini et al., 1996; Shiau and Chen, 2000; Lin et al., 2003; Stéphanie et al., 2010), and most studies on vitamin requirements have examined a single vitamin. However, assessments of vitamin combinations may yield a more realistic representation of vitamin requirements in animals, as appropriate combinations of VB2, VB6, niacin and pantothenic acid have been shown to improve the growth and meat quality of crucian carps (Lin et al., 2003). Tan et al. (2007) used an orthogonal design to evaluate the possible nutritional functions of vitamins A, D3, E, and C on gonadal development and the immune response of yearling eel. An orthogonal array design is a useful statistical tool for multi-factor analyses that can reflect a general condition with the fewest number of experimental trials and can be used to determine dominant contributing factors, as well as the appropriate combination of levels of each factor (Montgomery, 1991; Zheng and Jiang, 2003). Few experiments have been conducted to determine the vitamin requirements of fsh according to an orthogonal design (Rong et al., 1996; Lin et al., 2003). In previous studies, the recommended dietary VA, VB2and VB6requirements for the softshelled turtle were determined according to production experience, but limited information is available about the effects of these 3 vitamins on the growth of this reptile species.

The present study was conducted to explore the effects of vitamins on feeding, growth and protein utilization of juvenile soft-shelled turtles using an orthogonal experimental design. The fndings may aid in providing a basis to further optimize the vitamin supplementation in turtles’ diets.

2. Materials and Methods

2.1 Experimental designThe study was performed in a laboratory at Hebei Normal University, Shijiazhuang, Hebei Province, China. We used an OA933experimental design to study the effects of dietary supplementation of 3 vitamins at 3 levels (VA: 5000, 20,000 and 35,000 IU kg-1; VB2: 60, 120 and 180 mg kg-1; and VB6: 20, 70 and 120 mg kg-1) on the growth and development of softshelled turtles (Table 1). An orthogonal array design was used to determine which vitamin had the strongest effects on feeding, growth and protein utilization efficiency of soft-shelled turtles. In this experiment, 135 turtles were divided into 9 groups, which each included 15 individuals.

2.2 Experimental dietsVitamins A, B2and B6were added to the nine experimental diets (T1 to T9) as shown in Table 1. The main nutritional components of the basic experimental powder diets were measured (Table 2). To determine the nutrient digestibility, 0.1% chromium oxide, an inert marker, was added to each diet. The powder diets were blended with water (35%), formed into wet pellets and stored at –20°C.

2.3 Experimental animals and proceduresThe turtles were acclimated to the laboratory conditions for 3 weeks in 135 aquaria [60 cm (l) × 30 cm (w) × 30 cm (h), water volume of 20 L] and fed the T1 diet. Thewater temperature was maintained at 30±0.5°C using a thermostat-controlled electric heater. The photoperiod was maintained at 14L:10D, with illumination between 07:00 and 21:00. The pH ranged from 7.5 to 8.0, and the DO content was over 6 mg L-1.

Table 2 The ingredients and nutrient composition of the experimental diets.

We randomly allocated 135 turtles to the aquaria, with one turtle per aquarium. The average body weight of the turtles was 5.90±0.20 g (weight±SD). The turtles were fed their respective diets at a rate of 4% body weight per day twice daily at 08:00 and 16:00. Uneaten feed was collected, and feces were removed after 30 minutes of feeding and were then dried at 60°C to a constant weight. Approximately one-third of the water in each aquarium was exchanged every day to maintain the water quality. The experiment continued for 80 days.

2.4 Sample collection and measurementPrior to the experiment, 15 turtles were randomly collected for collecting the initial samples. At the end of the experiment, all turtles from each group were sampled. The protein contents of all turtle samples were measured. The diets, uneaten feed, feces and turtles were dried at 60°C to a constant weight and were then smashed and sieved using a sample sifter. The crude protein contents of the samples were determined using the Kjeldahl method, and their energy contents were measured using a calorimeter (DJL-9, Changsha Xingdian Instrument, Changsha, Hunan, China).

2.5 Data calculationThe survival rate(SR), feed intake (FI), specific growth rate (SGR), feed conversion ratio (FCR), apparent digestibility coefficient of dry matter (ADC), protein digestibility coefficient (PDC), protein effciency rate (PER), protein deposition rate (PDR) and energy effciency (EGE) were calculated as follows:

SR (%) = 100 × N2/ N1

FI (%) = 100 × F / [T (W1 + W2) / 2],

SGR (%d-1) = 100 (ln W2– ln W1) / T

FCR = F / (W2– W1)

ADC (%) = 1 – [(Cr2O3in diet / Cr2O3in feces) × 100%

PDC (%) = 1 – [(Cr2O3in diet / Cr2O3in feces) × (protein in feces / protein in diet)] × 100%

PER (%) = 100 (W2– W1) / Fp

PDR (%) = 100 × Bp/ Fp

EGE（%）= 100 × G / (C–F)

where N1and N2are the initial and final numbers of turtles in each tank, respectively; W1and W2are the initial and fnal body weights of the turtles (g), respectively; T is the duration of the experiment (d); F is the cumulative feed intake; Fpis the protein intake; and Bpis body protein gain.

G, C and F (kJ) are growth energy, intake energy, and faecal energy, respectively, in the energy budget equation (C = G + F + U + R); and C–F represent the energy assimilated by the turtles.

2.6 Data calculation and statistical analysesThe importance of the three vitamins for growth was evaluated based on the effectiveness of each vitamin according to calculated ranges (R) (Roy 1990) and the difference between the mean maximum and minimum values of each index at the three vitamin levels, which indicated the most infuential factor (i.e., the factor resulting in the greatest improvement) for growth performance (Yan et al., 2009).

The data were analyzed using Statistica 6.0 software (Statsoft Inc., Tulsa, OK, USA). One-way ANOVA was used to detect the differences among the treatment means at a 5% signifcance level, and Duncan’s multiple range test was used to evaluate the differences among the treatment means.

3. Results

3.1 Survival rate, feed intake and growthThere was no mortality during the 80 days of this experiment. The results revealed that the feed intake was the highest for the T3 diet, with signifcantly higher intake than the T5 or T6 diet (F = 1.46, df = 134, P3,5= 0.049, P3,6= 0.040) (Table 3). The feed intake ranges (R) for the three vitamins at the three levels varied from 0.038 to 0.083, and VA exhibited the largest range (Table 4). The order of importance of the vitamins to feed intake was VA＞VB2＞VB6, and the vitamin combination and levels resulting in the highest feed intake was A3, B23, and B63(Table 4).

There were no significant differences in the SGR among the treatments (F = 0.822, df = 134, P = 0.58). The SGR ranges (R) for the 3 vitamins varied from 4.8% to 18.4%, and VA exhibited the largest range. The order of importance of the vitamins to the SGR was VA＞VB2＞VB6, and the optimal vitamin combination for achieving the highest SGR was A3, B23, and B62(Table 4).3.2 Dietary nutrient utilizationThe FCR, PER, PDR, ADC and PDC are listed in Table 5. There were no significant differences in the ADC or PDC among the groups analyzed (FADC= 0.63, df = 134, PADC= 0.72; FPDC= 0.85, df = 134, PPDC= 0.92). The ADC ranges (R) varied from 0.25 to 1.25, and VA exhibited the largest range. The order of importance of the 3 vitamins to the ADC and PDC was VA＞VB2＞VB6, and the optimal vitamin combinations were A2, B22, and B63for the ADC and A2, B21, and B63for the PDC (Table 6).

During the experiment, no significant differences in the PER or FCR were detected among the nine treatment groups (FPER= 0.67, df = 134, PPER= 0.558; FFCR= 0.64, df = 134, PFCR= 0.74). The order of importance of the 3 vitamins to the PER and FCR was VA＞VB6＞VB2, and the optimal vitamin combination was A2, B22, and B61of vitamins for the PER and FCR (Tables 6 and Table 7).

T6 yielded a higher PDR than T1, T3 and T9 (F = 1.32, df = 134, P6,1= 0.049, P6,3= 0.035, P6,9= 0.043). The order of importance of the 3 vitamins to the PDR was VB2＞VA＞VB6, and the optimal vitamin combination was A2, B22, and B61for the PDR (Table 7).

3.3 Energy utilizationThere were signifcant differences in the energy intake among the nine groups (FEI= 1.06, df = 134, PEI= 0.041) (Table 8). Group T3 exhibited the greatest energy intake, which was significantly higher than those of groups T2, T5, and T6 (P3,2= 0.04, P3,5= 0.04, P3,6= 0.032). The energy intake ranges (R) for the three vitamins varied from 6.1 to 12.69, and VA exhibited the largest range. The order of importance of the vitamins with regard to energy intake was VA＞VB2＞VB6, and the vitamin combination resulting in the greatest energy intake was A1, B23, and B63(Table 9).

Significant differences in the energy efficiency for growth were also observed (FEGE= 1.06, df = 134, PEGE= 0.041); that of group T6 produced was greater than those of groups T1, T3, T4, T7, T8, and T9. The order of importance of the vitamins with regard to the energy effciency for growth was VB2＞VA＞VB6, and the vitamin combination resulting in the greatest energy effciency for growth was A2, B22, and B61(Table 9).

4. Discussion

Assessment of appropriate vitamin combinations may provide a more realistic representation of the vitamin requirements of animals, as appropriate combinations of VB2, VB6, niacin and pantothenic acid have been shown to improve the growth and meat quality of crucian carps (Lin et al., 2003). In the present study, no mortality, avitaminosis or hypervitaminosis was observed during the experiment, and the results indicated that dietary supplementation with the different combinations of VA, VB2and VB6did not significantly affect the SR of the soft-shelled turtles. The results also demonstrated that the vitamin combinations clearly affected the FI, PDR and EGE of the reptiles (P＜0.05).

In the present study, VA had much greater effects on the FI, SGR, ADC, PDC, FC and PER than VB2and VB6(Tables 4, 6 and 7), indicating that VA playsimportant roles in multiple processes, including those related to digestion, nutrient utilization and growth. VA supplementation at level 2 improved digestive functions (ADC, PDC, FC, PDR and PER) more than that at level 1 or 3. Further, VA supplementation at level 3 had greater effects on the SGR than that at the other levels, indicating that a high VA level (35,000 IU kg-1) can improve the feeding and growth rate of the softshelled turtles. The above results demonstrate that VA plays a broad and important role in juvenile turtle growth. Previous studies have suggested that the VA requirements of most finfish range from 1000 to 20,000 IU kg-1(Masumoto, 2002; Mohamed et al., 2003; Moren et al., 2004; Hernandez et al., 2005). Based on the appropriate levels, dietary VA supplementation at 20,000-35,000 IU kg-1should be used for soft-shelled turtles. The differing demands for VA between these two animals may be attributed to differences in metabolic processes (Chen and Huang, 2014). In contrast with the present study, the recommended dietary VA requirement for turtles was found to be 10800-11600 IU kg-1in the aforementioned study (Chen and Huang, 2014), and the turtle growth (WG, FCR and PER) in the present study was superior to that in this previous study. The discrepant results between

two studies may be due to the use of different ingredients, nutrient compositions and VA supplementation levels in the diets. In the present study, VA supplementation at level 3 (35,000 IU kg-1) had more benefcial effects on the turtle growth rate than that at level 2 (20,000 IU kg-1).

Table 3 Effects of the different diets on feeding and growth of Pelodiscus sinensis.

Table 4 Results of analysis of the effects of different vitamin levels on feed intake and growth.

Table 5 Effects of the different diets on diet utilization in Pelodiscus sinensis.

Table 6 Results of analysis of the effects of different vitamin levels on diet utilization.

Table 7 Results of analysis of the effects of different vitamin levels on PDR and PER.

Table 8 Effects of the different diets on energy intake and net energy effciency for growth.

Table 9 Results of analysis of the effects of different vitamin levels on energy intake and energy effciency for growth.

In this study, VB2had greater infuences on the PDR and EGE than VA and VB6based on the relative orders of importance of these vitamins, which is consistent with the fnding that the whole-body protein content in Jian carp increases with an increasing dietary ribofavin levels (Li et al., 2009). The results of this study indicate that VB2may play an important role in converting dietary protein and energy into usable protein and energy in the softshelled turtle. In previous studies, VB2at a suitable level has been shown to be conductive to the growth of some aquaculture animals (Xu et al., 1995; Souto et al., 2008; Li et al., 2010). Souto et al. (2008) have found that sea bream fed a VB2- enriched diet (17.7 mg kg-1) grew better than those fed a control diet (13.7mg kg-1). In addition, a low dietary VB2level (100 mg/kg) has been shown to result in a higher SGR than a high dietary VB2level (400mg kg-1) in shrimp (Xu et al., 1995), perhaps due to the high levels of digestive enzymes and energy necessary for separating VB2from proteins (Wang and Shan, 2007). In the present study, VB2supplementation at level 2 (120 mg kg-1) resulted in the optimal rates of absorption and conversion of protein and energy (Tables 7 and 9), and that at level 3 (180 mg kg-1) yielded an optimal growth rate compared with that at the other two levels; thus, the VB2level in the juvenile turtle diet should be approximately 120–180 mg kg-1.

Previous experiments have demonstrated that VB6infuences the PER and feed coeffcient ratio (FCR). The metabolism of this vitamin is related to dietary protein or amino acid metabolism in animals (Hilton, 1989; Giri et al., 1997). In the present study, VB6had fewer effects on protein metabolism than VA based on the order of importance of the vitamins (Tables 6 and 7). Further, VB6had a greater influence on the FCR than VB2, and the same result has been found in a study conducted by Lin et al. (2003) showing that VB6has important effects on digestive enzyme and alkaline phosphatase activities (He et al., 2009). The bass Lateolabrax japonicus and Jian carp Cyprinus carpio exhibit optimal growth atVB6concentratons of 20 mg kg-1(Zhong and Zhang, 2001) and 6.07mg kg-1(He et al., 2009), respectively. Further, the most appropriate VB6level for shrimp is approximately 140 mg kg-1(Xu et al., 1995). In the present study, based on the PER and FCR K values, VB6supplementation at level 1 (20 mg kg-1) was optimal compared with that at the other levels, and the PER and FCR gradually worsened with increasing VB6levels (Tables 6 and 7). In addition, VB6supplementation at level 2 (70 mg kg-1) resulted in a higher SGR of the turtles (Table 4). Therefore, VB6should be kept at a low level (20–70 mg kg-1) in the juvenile turtle diet.

The results of this study demonstrated that the order of importance of the 3 vitamins with regard to the turtle feed intake, growth and digestibility was VA＞VB2＞VB6and that the order of importance with regard to the conversion capacity was VB2＞VA＞VB6(Tables 4, 6, and 7). These findings suggest that at the levels tested, VA influenced feeding, growth, digestion and feed utilization, and had the strongest effects on the soft-shelled turtles, that VB2played an important role in growth effciency (PDR and EGE), and that VB6had greater effects on the FCR and PER than did VB2.

The results showed that the vitamin combination A2, B22, and B61generated the highest PDR and PER and that combination A3, B23, and B62resulted in optimal growth; thus, based on the growth results, the dietary VA, VB2and VB6requirements for soft-shelled turtles were estimated to be 35,000 IU kg-1, 180 mg kg-1and 70 mg kg-1, respectively.

AcknowledgementsThis work was fnancially supported by the National Natural Science Foundation of China (Nos. 30972261, 31172085, 31272315 and 41606137).

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*Corresponding authors: Dr. Zhencai YANG, from College of Life Science, Hebei Normal University, Shijiazhuang, Hebei Province, China, with his research focusing on reptiles nutrition.

E-mail: zcyang@mail.hebtu.edu.cn

Received: 26 January 2016 Accepted: 28 February 2016