The digestible phosphorus requirement in practical diet for largemouth bass (Micropterus salmoides) based on growth and feed utilization

Pu Wang , Xiaoqin Li , Zhen Xu , Dong Ji , Ming He , Jiangyu Dang ,Xiangjun Leng ,＊

a National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, 201306, China

b Centre for Research on Environmental Ecology and Fish Nutrition (CREEFN) of the Ministry of Agriculture, Shanghai Ocean University, Shanghai, 201306, China

c Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University, Shanghai, 201306, China

Keywords:

Largemouth bass

Digestible phosphorus

Growth

Feed utilization

A B S T R A C T

The present study was conducted to estimate the optimum requirement of phosphorus in practical diets for largemouth bass (Micropterus salmoides).Six iso-energetic and iso-nitrogenous diets were formulated with the supplementation of monocalcium phosphate (MCP) of 0, 5, 10, 15, 20 and 25 g/kg in basal diet containing 350 g/kg fish meal, respectively.The digestible phosphorus of the six diets was measured as 5.7, 6.7, 7.8, 8.7, 9.3 and 10.0 g/kg, respectively.Each diet was randomly fed to triplicate groups of 25 juvenile fish (initial body weight,16.0 ±0.2 g) for 60 days.The results showed that weight gain increased, and feed conversion ratio decreased with the increase of dietary phosphorus.When dietary MCP exceeded 15 g/kg (digestible phosphorus of 8.7 g/kg), the WG and FCR were maintained at the similar values.The crude ash, phosphorus and calcium contents in whole body, apparent digestibility of phosphorus and protein retention also increased with the increasing phosphorus level in diets.Vertebrae phosphorus and calcium levels and plasma phosphorus showed no further increase when dietary MCP reached 20, 20 and 10 g/kg with digestible phosphorus of 9.3, 9.3 and 7.8 g/kg,respectively.Broken-line analysis indicated that the digestible phosphorus requirement for largemouth bass were 8.9, 8.7 and 9.6 g/kg based on weight gain, feed conversion ratio and vertebral phosphorus content.In conclusion, the supplementation of MCP in a diet containing 350 g/kg fish meal improved the growth and feed utilization of largemouth bass, and the digestible phosphorus requirement was suggested to be 8.7-8.9 g/kg with MCP supplementation of 15.0-15.8 g/kg.

1.Introduction

Phosphorus is one of the major mineral elements in diet, which directly contributes to the formation of skeletal system, and it is also an important component of nucleus and membrane in animal cell (Watanabe, Satoh, & Takeuchi, 1988).The phosphorus deficiency in fish nutrition resulted in slow growth, low feed efficiency and poor bone mineralization (Sugiura, Hardy, & Roberts, 2004).Moreover, phosphorus deficiency also inhibited the esterification of free fatty acids to produce fatty acyl-CoA, reduced the oxidation of fatty acids, and increased gluconeogenesis and lipid deposition in the body (Roy & Lall,2003).However, Jokinen, Vielma, Aaltonen, and Koskela (2003) reported that phosphorus deficiency showed minor effects on the immune parameters in European white fish (Coregonus lavaretusL.), and the practical diet with sufficient phosphorus for normal growth did not compromise the immune function of this species.

Fish can absorb minerals directly from natural water (NRC, 2011),but the phosphorus content in fresh water and seawater is very low(Boyd, 1971; Lall, 2002), thus fish just absorbs very little phosphorus from the water (Phillips, Podoliak, Brockway, & Vaughn, 1958).Therefore, the required phosphorus of fish mainly comes from the diets.In grass carp (Ctenopharyngodon idella) (Liang, Liu, Tian, Yang, & Liang,2012), black seabream (Sparus macrocephalus) (Shao et al., 2008), large yellow croaker (Pseudosciaena croceaR.) (Mai et al., 2006) and Jian carp(Cyprinus carpiovar.Jian) (Xie et al., 2011), the dietary supplementation of inorganic phosphorus was reported to improve the weight gain (WG),reduce the feed conversion ratio (FCR), and the phosphorus requirement(available phosphorus) was also determined to be 8.43 g/kg, 5.4 g/kg,7.0 g/kg and 5.5 g/kg for these fishes, respectively.

Largemouth bass (Micropterus salmoides) is one of the commercially important freshwater fish in China, and has been widely cultured in recent years.In 2019, the aquaculture production of largemouth bass reached 477 808 tons in China (Ministry of agriculture and rural affairs of the people’s republic of China, 2020).At present, some studies have reported the protein requirement (Anderson, Kienholz, & Flickinger,1981; Breck, Wahl, & Hooe, 1996), suitable levels of dietary protein, fat(Chen et al., 2012) and digestible starch (Gou, Chen, Xu, Liu, & Yin,2015) for largemouth bass, but few was reported in phosphorus requirement.The only one report about the phosphorus requirement for largemouth bass we could find was the study of Shi et al.(2018).In this study, the WG and phosphorus retention showed the highest values when largemouth bass consumed a diet containing 5.1 g/kg digestible phosphorus (Shi et al., 2018).However, the basal diet contained a low phosphorus level due to a low fish meal inclusion (180 g/kg), which was much lower than that in the commercial diet, and such a result could not provide practical information for the feed production.

In practical production, the commercial diets for largemouth bass usually contain about 350-550 g/kg fish meal, which contains a plenty of phosphorus.However, the accurate inclusion of inorganic phosphorus is still unclear in practical diets for largemouth bass.Thus, to determine a suitable supplementation of inorganic phosphorus in practical diet is a crucial parameter to design an effective and environment-friendly diet without excessive phosphorus emission.Therefore, in the present study,graded levels of monocalcium phosphate (MCP) were supplemented into basal diet to determine the optimal digestible phosphorus requirement for largemouth bass based on growth, body composition, feed utilization and phosphorus contents in vertebrae and plasma.

2.Materials and methods

2.1.Ethical statement

All animal tests were approved by the Animal Ethics Committee of Shanghai Ocean University (Shanghai, China), and complied with the“Guidelines on Ethical Treatment of Experimental Animals” formulated by the Ministry of Science and Technology, China.

2.2.Experimental diets

Six practical diets were formulated to contain graded available phosphorus levels of 10.9, 12.0, 13.2, 14.3, 15.4 and 16.5 g/kg, with the MCP supplementation of 0, 5, 10, 15, 20 and 25 g/kg, referring as P0,P5, P10, P15, P20, P25, respectively.The contents of crude protein (CP)and crude lipid (CL) in all diets were designed to be about 470 g/kg and 120 g/kg, respectively.Yttrium trioxide (Y2O3) was included in diets at 0.5 g/kg as an indicator for digestibility analysis.The protein ingredients were ground and sieved (60 mesh), then mixed with oil and water carefully.A single screw extruder (SLP-45; China Fisheries Machinery Research Institute, Shanghai, China) was used to make sinking pellets with a diameter of 2.0 mm at a pelleting temperature of 85 ±5 ℃.After the air-drying, all diets were stored at 4 ℃ for later use.

The diet formula and the proximate composition are shown in Table 1.The digestible phosphorus levels were calculated based on the following digestibility test.

Table 1Feed formulation and proximate composition of experimental diets, g/kg.

2.3.Experimental fish and feeding management

Juvenile largemouth bass were obtained from Qingpu Aquaculture Station, Shanghai, China.Before the feeding trial, all fish were stocked in indoor pools and fed the commercial diet containing 490 g/kg CP and 90 g/kg CL (Zhejiang Yuehai Feed Company, China) for 4 weeks to acclimate to laboratory conditions at Binghaifish feeding station of Shanghai Ocean University.Four hundred and fifty fish with an average initial weight of 16.0 ±0.2 g were randomly allotted into 18 cages (1.3m × 1.2 m × 1.1 m) hung in indoor pools with triplicates (25 fish/cage).During the feeding trial, all fish were manually fed with one of the six diets twice a day (8:00, 16:00) with a daily feed intake of 3%-5% of body weight for 60 days.Proper adjustment was conducted according to the ingestion and the weather to ensure that all the fish could consume the diet immediately, and all cages were kept with the similar amount of feed intake.During the feeding period, the water temperature, and dissolved oxygen were 26-30 ℃, and 5.0-6.1 mg/L.The pH ranged from 7.2 to 7.9 mg/L, and total ammonia was less than 0.2 mg/L.

2.4.Sample collection and analysis

At the end of the feeding trial, all fish were deprived of diets for 24 h.The number of fish per cage was counted and the fish were weighed to calculate survival, WG and FCR.Six fish were randomly selected from each cage and sampled after MS-222 anesthesia.Two fish were used for proximate composition analysis of whole body, and the other four fish were recorded body weight and body length, then blood was taken from the tail vein with a 1% heparin-soaked needle.The blood samples were centrifuged for 10 min (3000 rpm), and serum was collected and stored at -80 ℃ for future use.Then, the fish were dissected and the visceral weight, liver weight and intraperitoneal fat weight were measured immediately to calculate condition factor (CF), intraperitoneal fat ratio(IFR), viscerosomatic index (VSI) and hepatosomatic index (HSI).The muscles above the lateral line from both sides of the fish were sampled and stored at -20 ℃ for chemical composition and phosphorus analyses.According to the method of Shao et al.(2008), the spinal column was collected after being cooked in microwave for 60-80s, then dried at 105 ℃, and stored at -20 ℃ for the determination of calcium and phosphorus contents.

2.4.1.Growth and body index

WG (%) =100 ×weight gain (g)/initial body weight (g)

FCR =feed intake (g)/weight gain (g)

CF (g/cm3) =100 ×body weight (g)/body length (cm)3

Survival (%) =100 × final number of fish/initial number of fish

IFR (%) =100 ×intraperitoneal fat weight (g)/body weight (g)

VSI (%) =100 ×visceral weight (g)/body weight (g)

HSI (%) =100 ×hepatopancreas weight (g)/body weight (g)

2.4.2.Proximate composition of diets, feces and whole fish

The analysis of proximate composition referred to the method of AOAC (1995).Moisture was analyzed by an oven at 105 ℃, and crude protein (CP) was determined by Auto Kjeldahl System (2300-Auto--analyzer, Foss Tecator, Sweden).Crude lipid (CL) was measured by the method of chloroform-methanol.Ash content was analyzed by combustion at 550 ℃ (SXL-1008 Muffle furnace, Shanghai Jinghong Experimental Equipment Company, China).The calcium and phosphorus contents in whole fish, tissues, diets and feces were determined by calcium methyl thymol blue and phosphomolybdic acid kits from Nanjing Jiancheng Institute of Bioengineering (Nanjing, China).Nutrient retention was calculated with the following formula:

In the above equation,W0andWtwere the initial weight (g) and final weight (g),respectively;W0nandWtnwere the nutrient content in whole body of initial fish and final fish, respectively;Wfwas the amount of feed intake;Wfnwas nutrient content in diet, where n represents protein, fat,ash and phosphorus, respectively.

2.4.3.Apparent digestibility of nutrients

In the last two weeks of the feeding trial, feces were collected by siphoning after 2 h of the second meal every day.Feces samples were dried at 60 ℃ and then preserved at -20 ℃ to analyze nutrient digestibility.The yttrium content was determined with plasma emission spectroscopy (ICP) (Vista MPX; VARIAN, Alo Alto, CA, USA).The Apparent digestibility coefficients (ADC) of nutrients (CP, CL and phosphorus) were calculated according to the following formulas:

In the above equations, a and b represent Yttrium content in feces and diet, c and d represent nutrients content in feces and diet.

2.5.Statistical analysis

All data were expressed as mean ±standard deviation (SD), and SPSS 17.0 statistical software (Statistical Package for the Social Sciences) was used to perform one factor analysis of variance.If significant differences were detected, the Tukey multiple range test was used for determining the statistical significance among groups.Mean values were considered significantly different if the P value was less than 0.05.Dietary phosphorus requirement was estimated by broken-line regression analysis(Robbins et al.1979; Robbins 1986).

3.Results

3.1.Growth performance

As shown in Table 2, there was no significant difference in survival,CF and IFR among all the groups (P ＞0.05).The WG increased, and FCR,HSI and VSI decreased with the increasing level of dietary phosphorus(P ＜0.05).When dietary MCP increased from 15 g/kg to 25 g/kg, the WG and FCR showed no significant difference among the three groups.In P0 group, two deformed fish were observed with a small body and bended caudal vertebrae, accounting for 2.7% of the total.

Table 2Effects of dietary phosphorus on growth and body morphometric indices of largemouth bass.

Broken-line analysis showed that the requirement of digestible phosphorus for largemouth bass was 8.9, 8.7 g/kg based on WG and FCR(Figs.1 and 2).Correspondingly, the MCP supplementation was 15.8,15.0 g/kg at this point.

Fig.1.Relationship between weight gain and dietary digestible phosphorus level.

Fig.2.Relationship between feed conversion ratio and dietary digestible phosphorus level.

3.2.Body and muscle composition

As shown in Table 3, CL in whole body and muscle significantly decreased, and ash, phosphorus and calcium contents in whole body increased with the increasing phosphorus level (P ＜0.05).There were no significant differences in proximate composition in muscle among all the groups (P ＞0.05) except CL.

Table 3Effect of dietary phosphorus on whole body and muscle composition of largemouth bass, g/kg wet weight.

3.3.Nutrient utilization

As shown in Table 4, the apparent digestibility of dry matter(ADCDM) and phosphorus (ADCP), the retentions of ash and protein increased with the increasing level of dietary phosphorus.When dietary digestible phosphorus reached 8.7 g/kg, these indicators showed no significant difference among the groups of P15, P20 and P25 (P ＞0.05).While lipid retention decreased with the increasing dietary phosphorus(P ＜0.05).P15 group presented the highest phosphorus retention, and no significant difference was found in ADCCPand ADCCLamong all the groups (P ＞0.05).

Table 4Effects of dietary phosphorus on nutrient digestibility and retention of largemouth bass, %.

3.4.Calcium and phosphorus contents in vertebral and plasma

As shown in Table 5, the contents of phosphorus and calcium in vertebrate and phosphorus in plasma increased with the increasing level of dietary phosphorus (P ＜0.05), and vertebrae phosphorus and calcium contents kept stable when dietary digestible phosphorus reached 9.3 g/kg (P ＞0.05).There were no significant differences in the ratio of calcium to phosphorus in vertebrae and plasma calcium content among all the groups (P ＞0.05).

Table 5Effect of dietary phosphorus on calcium and phosphorus contents in vertebrae and plasma of largemouth bass.

Broken-line analysis showed that the requirement of digestible phosphorus for maintaining maximum phosphorus storage was 9.6 g/kg based on the vertebrae phosphorus content (Fig.3).

Fig.3.Relationship between vertebrae phosphorus content and dietary digestible phosphorus level.

4.Discussions

In fish, the phosphorus deficiency usually causes the reduced growth and feed utilization, the deformed skeleton and lower ash level in the body (Borlongan & Satoh, 2001).In addition, the disintegration and necrosis of intestine, liver, pancreas, kidney, spleen, heart and parotid cells were also observed in juvenile Jian carp fed diets deficient in phosphorus (Xie et al., 2011).When dietary available phosphorus reached 7.7 g/kg, 10.2 g/kg and 11.2 g/kg, European sea bass (Dicentrarchus labraxL.) (Olivateles & Pimentelrodrigues, 2004), haddock(Melanogrammus aeglefinus,L.) (Roy & Lall, 2003) and gibel carp (Carassius auratus gibelio) (Zhang, Xu, Li, & Leng, 2016) displayed a good growth performance.In the present study, P0 group (no MCP supplementation) also showed a slow growth, and two fish were observed the skeletal deformity, which may be caused by dietary phosphorus de ficiency.The response of WG and FCR to dietary digestible phosphorus level was agreement with broken-line model, and the requirement of digestible phosphorus for largemouth bass was estimated to be 8.7-8.9 g/kg.

The present study indicated that the VSI and HSI rather than CF and IFR, decreased with the increasing level of dietary phosphorus.Roy and Lall (2003) also found that dietary phosphorus did not affect the CF of juvenile haddock, but HSI increased and then decreased with the increasing dietary phosphorus.In juvenile black seabream, HSI presented a negative correlation with dietary phosphorus level (Shao et al.,2008).This may be the result of lipid accumulation in liver due to the deficiency of phosphorus.

Xie et al.(2011) found that lipid content in whole body of Jian carp negatively correlated with phosphorus content in diet.The similar findings were also reported in the studies of black seabream (Shao et al.,2008), grass carp (Liang et al., 2012), haddock (Roy & Lall, 2003) and Japanese flounder (Paralichthys olivaceus) (Uyan, Koshio, Ishikawa,Uyan, Ren, Yokoyama, & Komilus, 2007).Such results may be contributed to the fact that high dietary phosphorus enhanced the β-oxidation of fatty acid, thus reducing the lipid deposition.On the contrary, insufficient dietary phosphorus inhibited the formation of acetyl-CoA, and the oxidative combustion of fatty acids and fat utilization were suppressed, resulting in the lipid accumulation in fish(Roy & Lall, 2003).In this study, the increasing dietary phosphorus reduced whole-body lipid and lipid retention, but increased protein retention, which indicated that sufficient phosphorus promoted the utilization of lipid, and more protein was used for the growth.The study of Yang, Lin, Liu, and Liou (2006) had proved that the supplementation of phosphorus in diet reduced the excretion of ammonia.

In fish nutrition, whole body ash and phosphorus levels were often used as indicators reflecting phosphorus status (Hardy, Fairgrieve, &Scott, 1991; Mai et al., 2006; Skonberg, Yogev, Hardy, & Dong, 1997).It has been reported that the contents of ash, calcium and phosphorus in whole body increased with the increasing phosphorus level in the diet of grass carp (Liang et al., 2012), Jian carp (Xie et al., 2011), juvenile milkfish (Chanos) (Borlongan & Satoh, 2001) and black seabream (Shao et al., 2008).In the present study, as dietary phosphorus increased, the contents of ash, phosphorus and calcium in whole body, and the retentions of ash and phosphorus also increased, but the contents of ash,calcium and phosphorus in muscle showed no significant difference among all the groups.Maybe bone is the main deposition part of minerals, while muscle contains less calcium and phosphorus, thus, muscle is not sensitive to the change of dietary phosphorus.

Compared to the weight gain, the phosphorus digestibility can directly reflect the utilization of phosphorus in diet (Liu, 2010).In this study, the phosphorus digestibility increased with the increasing digestible phosphorus from 5.7 to 8.7 g/kg, and then remained stable regardless of the further increase of dietary phosphorus.With the increase of MCP supplementation from 0 to 15.5 g/kg, the phosphorus digestibility of largemouth bass increased from 42.8% to 52.7% (Shi et al., 2018).In Jian carp, phosphorus digestibility also increased with the increasing phosphorus level (Qin, Yang, Leng, Wu, & Li, 2015, pp.383-390).MCP has a good solubility with a high digestibility, thus the increased phosphorus digestibility may be attributed to the increasing supplementation of MCP.

The fish bone is the main deposition part of phosphorus, and it is also involved in the phosphorus metabolism.Therefore, vertebrae phosphorus is usually used as an indicator to evaluate phosphorus demand.The present study showed that the contents of phosphorus and calcium in vertebrae increased with the increasing digestible phosphorus level until it reached 9.3 g/kg (P20 group).Shao et al.(2008) found that the supplemental phosphorus significantly increased ash, calcium and phosphorus contents in vertebrae of black seabream.In milkfish, the phosphorus and calcium contents in vertebra increased with the total phosphorus increasing from 2.8 (without P supplementation) to 8.8 g/kg(Borlongan & Satoh, 2001).In this study, broken-line analysis based on vertebra phosphorus indicated that the optimum digestible phosphorus requirement for largemouth bass was 9.6 g/kg, which was higher than the value based on WG (8.9 g/kg).The similar results were also reported in snakehead (Channa argus×Channa maculata) (Shen, Chen, Chen, Lin,Chen, Zhang, & Luo L), sunshine bass (Morone chrysops♀ ×M.saxatilis♂) (Brown, Jaramillojr, & Gatliniii, 1993), large yellow croaker (Mai et al., 2006), blue tilapia (Oreochromis aureus) (Robinson, Labomascus,Brown, & Linton, 1987) and rainbow trout (Ketola & Richmond, 1994).The above results showed that vertebrae have a strong buffering capacity responding to the change of dietary phosphorus.The phosphorus deposition in skeletal will not maximize when the fish reach the highest growth.

Most of the calcium, phosphorus and other minerals were stored in the bones of fish, and constantly involved in the substances exchange with blood and extracellular fluids (Lall, 2002).Sometimes, the plasma phosphorus was used to reflect the phosphorus status in the body (Zhang et al., 2006).In the present study, plasma phosphorus increased with the increasing phosphorus level, and reached the plateau when digestible phosphorus exceeded 7.8 g/kg.The studies on haddock (Roy & Lall,2003), Japanese seabass (Lateolabrax japonicus) (Zhang et al., 2006),juvenile silver perch (Bidyanus bidyanus) (Yang et al., 2006), and snakehead (Shen et al., 2016) presented the similar results.However, no significant difference in serum phosphorus content was observed in hybrid sturgeon (♀Huso huso· × ♂Acipenser schrenckii) when the fish were fed diets containing 6.3 g/kg and 21.2 g/kg available phosphorus(Jin, Wang, Tang, Xie, & Dai, 2012), which indicated that serum phosphorus would remain stable when the phosphorus demand was met.Besides the effect of dietary phosphorus, serum phosphorus concentration also depended on the sampling time (Rodehutscord, 1996).Thus,serum phosphorus, and other indicators, should be comprehensively considered to determine the phosphorus status in the body.Unlike serum phosphorus, serum calcium level was not affected by the supplementation of phosphorus in diets (Table 5), which was in line with the studies in snakehead (Shen et al., 2016), rainbow trout (Ketola &Richmond, 1994), large yellow croaker (Mai et al., 2006), and silver perch (Yang et al., 2006).The reason is that fishes can directly absorb sufficient calcium from the water regardless of the dietary calcium and phosphorus.

5.Conclusion

In the present study, the supplementation of MCP in a practical diet containing 350 g/kg fish meal improved the growth and feed utilization of largemouth bass.The requirement for digestible phosphorus was estimated to be 8.7-8.9 g/kg based on weight gain and feed conversion ratio from the broken-line model, and the MCP supplementation was 15.0-15.8 g/kg at this point.

CRediT authorship contribution statement

Pu Wang: Data curation, Formal analysis, Writing - original draft,Writing - review & editing, handled feeding trial, laboratory works, data analysis, writing and editing original draft.Xiaoqin Li: Conceptualization, Supervision, Writing - review & editing, Funding acquisition,contributed funding supports, conceptualization, supervision, reviewing and editing manuscript.Zhen Xu: supported diets preparation,fish feeding and laboratory works.Dong Ji: supported diets preparation,fish feeding and laboratory works.Ming He: supported diets preparation,fish feeding and laboratory works.JiangYu Dang: supported diets preparation,fish feeding and laboratory works.Xiang-jun Leng:Conceptualization, Supervision, Writing - review & editing, Funding acquisition, contributed funding supports, conceptualization, supervision, reviewing and editing manuscript.

Declaration of competing interest

We declare that we do not have any financial and personal relationships with other people or organizations that may improperly affect our work, and there are no professional or other individuals of any nature or kind in any product, service and/or company benefits can be interpreted as having an impact on the content of this article.

Acknowledgment

This study was supported by Research and Supporting Platform of Efficient Aquaculture (A1-3201-19-3003).